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Full text of "Clinical diagnosis; a manual of laboratory methods"

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Presented by 
Estate of Harry W, Forbes, D.O. 





E COLLEGE OF OSTEOPATHIC PHYSICIANS 
AND SURGEONS • LOS ANGELES, CALIFORNIA 



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UNIVERSITY OF CAMPORNIA 

CALIFORNIA COLLEGE OF MEDICINE 

LIBRARY 

JlIN 1 1^71 

IRVINE, CALIFORNIA 92664 



Digitized by tlie Internet Arcliive 

in 2007 witli funding from 

IVIicrosoft Corporation 



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Scale of urinary colors, according to \'of;i-l. 



Clinical Diagnosis 

A MANUAL OF LABORATORY METHODS 



BY 

JAMES CAMPBELL TODD. Ph. B.. M. D. 

PROFESSOR OF PATHOLOGY, UNIVERSITY OF COLORADO 



Illustrated 



Second Edition, Revised and Enlarged 



PHILADELPHIA AND LONDON 

W. B. SAUNDERS COMPANY 

1912 



Copyright, 1908, by W. B. Saunders Company. Revised, reprinted, and 
recopyrighted January, 1912 



Copyright, 1912, by W. B. Saunders Company 



PRINTED IN AMERICA 

PRESS OF 

W. B. SAUNDERS COMPANV 

PHILADELPHIA 



TO 

MY FATHER 

THESE PAGES ARE 
AFFECTIONATELY DEDICATED 



'> 



•<\ 



PREFACE TO THE SECOND EDITION 




While the original purpose of this book — to present 

^ clearly and concisely the various laboratory methods 

»^AJ which are of use in clinical medicine — has not been lost 

^ sight of, its scope has been somewhat enlarged in the 

oX present edition. 

^ Each section has been carefully revised and much new 

'^^ material has been added to every chapter. Among the 

'0 many additions may be mentioned: the use of artificial 

>v light and the importance of numerical aperture in micro- 

'S^ ^copic work; photomicrography with simple apparatus; 

S* the antif ormin method for tubercle bacilli ; detection and 

X significance of albumin in the sputum; Tsuchiya's modi- 

I fication of Esbach's test; the formalin test for ammonia 

and Benedict's methods for sugar in urine; volume index 

Ja of red blood-corpuscles; Wright and Kinnicutt's method 

i^ of counting blood-platelets; Harlow's blood-stain; a sim- 

"'^ pie technic for the diagnosis of typhoid fever by blood- 

_J cultures; the Wassermann reaction, and Frothingham's 

iaX impression method in the diagnosis of rabies. 

Because of the growing importance of animal parasites, 
this chapter has been entirely rewritten and more than 
doubled in extent. Two new chapters have been added: 
.one upon Bacteriologic Methods, which supplements the 
methods given in other portions of the book, and one 
upon Preparation and Use of Vaccines, including thera- 
peutic and diagnostic use of tubercuHn. 

22865 



lO PREFACE 

Some of the illustrations have been replaced with better 
ones and many new pictures have been added, including 
eight photomicrographs by Dr. W. P. Harlow, and a con- 
siderable number by the author. Through courtesy of 
Dr. Langdon Frothingham, of Harvard University, a 
colored plate showing Negri bodies as seen in his im- 
pression method has been included. 

The author wishes to express his indebtedness to Fran- 
cis Ramaley, Ph.D., Professor of Biology in the Univer- 
sity of Colorado, and T. D. A. Cockerell, Professor of 
Systematic Zoology, for suggestions as to the nomen- 
clature of animal parasites; and to Dr. A. R. Peebles, 
Professor of Medicine, for suggestions and aid through- 
out the revision. 

J. C. T. 
Boulder, Colorado. 



PREFACE 



This book aims to present a clear and concise state- 
ment of the more important laboratory methods which 
have clinical value, and a brief guide to interpretation 
of results. It is designed for the student and practi- 
tioner, not for the trained laboratory worker. It had 
its origin some years ago in a short set of notes which 
the author dictated to his classes, and has gradually 
grown by the addition each year of such matter as the 
year's teaching suggested. The eagerness and care with 
which the students and some practitioners took these 
notes and used them convinced the writer of the need 
of a volume of this scope. 

The methods oflfered are practical; and as far as 
possible are those which require the least complicated 
apparatus and the least expenditure of time. Simplicity 
has been considered to be more essential than absolute 
accuracy. Although in many places the reader is given 
the choice of several methods to the same end, the 
author believes it better to learn one method well than 
to learn several only partially. 

More can be learned from a good picture than from 
any description, hence especial attention has been given 
to the illustrations, and it is hoped that they will serve 
truly to illustrate. Practically all the microscopic struc- 



12 PREFACE 

tures mentioned, all apparatus not in general use, and 
many of the color reactions are shown in the pictures. 

Although no credit is given in the text, the recent 
medical periodicals and the various standard works have 
been freely consulted. Among authors whose writings 
have been especially helpful may be mentioned v. Jaksch, 
Boston, Simon, Wood, Emerson, Purdy, Ogden, Ewald, 
Ehrlich and Lazarus, Da Costa, Cabot, Osier, Stengel, 
and McFarland. 

The author wishes hereby to express his indebtedness 
to Dr. J. A. \\'ilder. Professor of Pathology in the Den- 
ver and Gross College of Medicine, for aid in the final 
revision of the manuscript; and to W. D. Engel, Ph.D., 
Professor of Chemistry, for suggestions in regard to de- 
tection of drugs in the urine. He desires to acknowl- 
edge the care with which Mr. Ira D. Cassidy has made 
the original drawings, and also the uniform courtesy of 
W. B. Saunders Company during the preparation of 
the book. 

J. C. T. 

Denver, Colorado. 



CONTENTS 



INTRODUCTION page 

Use of the Microscope 17 

CHAPTER I 

The Sputum 36 

Physical Examination 38 

Microscopic Examination 40 

Unstained Sputum 40 

Stained Sputum 48 

Chemic Examination 63 

Sputum in Disease 64 

CHAPTER II 

The Urine 68 

Physical Examination 70 

Chemic Examination 80 

Normal Constituents 80 

Abnormal Constituents 99 

Microscopic Examination 138 

Unorganized Sediments 141 

Organized Sediments 151 

Extraneous Structures 171 

The Urine in Disease 173 

CHAPTER III 

The Blood 180 

Hemoglobin 184 

Enumeration of Erythrocytes 192 

Color Index 200 

Volume Index 200 

Enumeration of Leukocytes 202 

Decrease in Number of Leukocytes 202 

Increase in Number of Leukocytes 203 

Leukocytosis 203 

Leukemia 208 

Method of Counting Leukocytes 209 

13 



14 CONTENTS 

PAGE 

Enumeration of Blood-plaques 213 

Study of Stained Blood 216 

Making and Staining Blood-films 216 

Study of Stained Films 225 

Blood Parasites 244 

Bacteria 244 

Animal Parasites 247 

Serum Reactions 257 

Tests for Recognition of Blood 274 

Special Blood Pathology 275 

Anemia 275 

Leukemia 280 

CHAPTER IV 

The Stomach 284 

Examination of the Gastric Contents 284 

Obtaining the Contents 285 

Physical Examination 288 

Chemic Examination 289 

Microscopic Examination 301 

The Gastric Contents in Disease 304 

Additional Examinations which Give Information as to the 

Condition of the Stomach 306 

CHAPTER V 

The Feces 310 

Macroscopic Examination 311 

Chemic Examination 314 

Microscopic Examination 315 

Functional Tests 320 

CHAPTER VI 
Animal Parasites 323 

Protozoa 326 

Sarcodina 328 

Mastigophora (Flagellata) 330 

Sp)orozoa 338 

Infusoria 339 

Vermidea , 340 

Platyhelminthes 341 

Nemathelminthes 353 

Arthropoda 366 



CONTENTS 15 

CHAPTER VII 

Miscellaneous Examinations 367 

Pus 367 

Peritoneal, Pleural, and Pericardial Fluids 371 

Cerebrospinal Fluid 374 

Animal Inoculation 375 

The Mouth 377 

The Eye 381 

The Ear 383 

Parasitic Diseases of the Skin 384 

Milk 384 

Syphilitic Material 388 

Semen , 391 

Diagnosis of Rabies 393 

CHAPTER VIII 

Bacteriologic Methods 396 

Apparatus 396 

Sterilization • 399 

Preparation of Culture-tubes 400 

Culture-media 401 

Staining Methods 407 

Methods of Studying Bacteria 412 

Characteristics of Special Bacteria 415 

CHAPTER IX 

Preparation and Use of Vaccines 419 

Preparation of Vaccine 419 

Method of Use 425 

Dosage 425 

Therapeutic Indications 426 

Tuberculins 428 

Tuberculin in Diagnosis 429 

APPENDIX 

Apparatus, Reagents and Stains 432 

Apparatus 432 

Reagents and Stains 434 

Weights, Measures, etc., with Equivalents 439 

Temperature 440 

Index 441 



CLINICAL DIAGNOSIS 



INTRODUCTION 
USE OF THE MICROSCOPE 

There is probably no 
laboratory instrument 
whose usefulness de- 
pends so much upon 
proper manipulation as 
the microscope, and 
none is so frequently 
misused by beginners. 
Some suggestions as to 
its proper use are, there- 
fore, given at this place. 
It is presumed that the 
reader is already famil- 
iar with its general con- 
struction (Fig. i). 

For those who wish 
to understand the prin- 
ciples of the microscope 
and its manipulation — 
and best results are im- 
possible without such an 
understanding — a care- 
ful study of some stan- 

2 




Fig. I. — Handle-arm microscope: E, Eye- 
piece; D, draw-tube; T, body-tube; RN, 
revolving nose-piece; O, objective; PH, pinion 
head; MH, micrometer head; HA, handle-arm; 
SS, substage; S, stage; M. mirror; B, base; R, 
rack; P, pillar; I, inclination joint. 
17 



l8 INTRODUCTION 

dard work upon microscopy, such as those of Carpenter, 
Spitta, and Sir A. E. Wright, is earnestly recommended. 
It is also recommended that the beginner provide him- 
self with some slides of diatoms, for example, Pleuro- 
sigma angulalum, Surirella gemma, and Amphipleura 
pellucida, costing fifty cents each. Faithful practice 
upon such test-objects, in the light of the principles of 
microscopy, will enable the student to reach, intelli- 
gently, an accuracy in manipulation to which the ordi- 
nary laboratory worker attains only slowly and by rule 
of thumb. He will soon find that the bringing of an 
object into accurate focus is by no means all of micros- 
copy. 

Illumination. — Good work cannot be done without 
proper illumination. It is difficult to lay too much 
stress upon this point. 

The light which is generally recommended as best is 
that from a white cloud, the microscope being placed 
by preference at a north window, to avoid direct sun- 
light. Such light is satisfactory for all ordinary work. 
Artificial light is, however, imperative for those who must 
work at night, and is a great convenience at all times. 
Properly regulated artificial light, moreover, offers 
decided advantages over daylight for critical work. 
Almost any strong light which is diffused through a 
frosted globe will give fair results. The inverted Wels- 
bach light with such a globe is excellent. The follow- 
ing plan is much used abroad, and gives results equal 
to the best daylight: A Welsbach lamp or strong elec- 
tric light is used, and a glass globe — a six-inch round- 
bottom flask answers admirably — is placed between it 
and the microscope, to act as a condenser (Fig. 2). 



USE OF THE MICROSCOPE 



19 



The flask should be at a distance equal to its diameter 
from both the Hght and the mirror of the microscope. 
In order to filter out the yellow rays the flask is filled 
with water to which have been added a few crystals 
of copper sulphate and a little ammonia. 

For critical work, the method suggested by Sir A. E. 
Wright is to be preferred. He has shown that fog is 
dispelled and definition is Improved if the size of the 
Hght source is so regulated that its image, thrown upon 





Fig. 2. — Illumination with water-bottle condenser. 



the slide by the condenser, coincides with the real field 
of the objective. Upon this principle a very neat and 
satisfactory microscope lamp, shown in Fig. 3, has been 
designed by B. H. Matthews. It is fitted with iris- 
diaphragm, condensing lens, small electric light, and 
reflector, and has a slot in which a ray filter or ground- 
glass disc may be inserted. 

Illimiination may be either central or oblique. Central 
illumination is to be used for all routine work. To ob- 



20 



INTRODUCTION 



tain this, the mirror should be so adjusted that the light 
from the source selected is reflected directly up the tube 
of the microscope. This is easily done by removing the 
eye-piece and looking down the tube while adjusting the 
mirror. The eye-piece is then replaced, and the light 
reduced as much as desired by means of the diaphragm. 
With daylight, it is best to use the plane mirror; with 
artificial light, the concave mirror. 




Fig- 3- — Matthews' microscope lamp with iris-diaphragm. 

Obhque illumination is to be used only to bring out 
certain structures more clearly after viewing them by 
central light: as, for example, to show the edges of a 
hyahne cast by throwing one of its sides into shadow. 
Oblique illumination is obtained in the more simple 
instruments by swinging the mirror to one side, so that 
the light enters the microscope obliquely. The more 
complicated instruments obtain it by means of a rack 



USE OF THE MICROSCOPE 21 

and pinion, which moves the diaphragm laterally. 
Beginners frequently use oblique illumination without 
recognizing it, and are thereby much confused. If the 
light be oblique, an object in the center of the field will 
appear to move from side to side when the fine adjust- 
ment is turned back and forth. 

The amount of light is even more important than its 
direction. It is regulated by the diaphragm. It is 
always best to use the least light that will show the object 




a b 

Fig. 4. — a, Hyaline casts, one containing renal cells; properly subdued illumination; 
b, same as a; strong illumination. The casts are lost in the glare, and only the renal 
cells are seen. (From Greene's "Medical Diagnosis"). 

well. Unstained objects require very subdued light. 
Beginners constantly use it too strong. Strong light will 
often render semitransparent structures, as hyaline casts, 
entirely invisible (Fig. 4). Stained objects, especially 
bacteria, require much greater light. 

Dark Ground Illumination. — This consists in cutting 
out the central rays of light and directing the peripheral 
rays against the object from the side. Only those rays 
which strike the object and are reflected pass into the 
objective. The object is bright upon a black back- 



22 INTRODUCTION 

ground. By means of this form of illumination very 
minute structures can be seen, just as particles of dust 
in the atmosphere become visible when a ray of sunlight 
enters a darkened room. 

Dark ground illumination for low-power work can be 
obtained by means of the ring stops with central discs 
which accompany most microscopes when purchased. 
The stop is placed in a special ring beneath the con- 
denser. By varying the size of the central disc good 
results can be had with the lower power dry lenses. 

For oil-immersion work a special condenser is neces- 
sary. With some makes it is placed upon the stage of 
the microscope; with others it is substituted for the 
regular condenser. It requires an intense light, like 
direct sunlight or the Liliput arc-light. 

The chief use of dark ground illumination in cHnical 
work is for demonstration of Treponema pallidum in 
fresh material (Fig. 158). 

The Condenser. — For the work of the clinical labora- 
tory a substage condenser is a necessity. Its purpose 
is to condense the light upon the object to be examined. 
For critical work the light must be focused on the object 
by raising or lowering the condenser by means of the 
screw provided for the purpose. The image of the light 
source will then appear in the plane of the object. This 
is best seen by using a low-power objective and ocular. 
Should the image of the window-frame or other nearby 
object appear in the field and prove annoying, the con- 
denser may be raised or lowered a little. It is often 
advised to remove the condenser for certain kinds of 
work, but this is not necessary and is seldom desirable. 

It is very important that the condenser be accurately 



USE OF THE MICROSCOPE 23 

centered, and most high-grade instruments have center- 
ing screws by which it can be adjusted at any time. The 
simplest way to recognize whether the condenser is 
centered is to close the diaphragm beneath it to as small 
an opening as possible, then remove the eye-piece and 
look down the tube. If the diaphragm opening does 
not appear in the center of the field, the condenser is out 
of center. 

The use of the condenser is further discussed in the 
following section. 

Objectives and Eye-pieces. — Unfortunately, different 
makers use different systems of designating their lenses. 
The best system, and the one chiefly used in this country, 
is to designate objectives by their focal lengths in milli- 
meters, and eye-pieces by their magnifying power, 
indicated by an " X ." Most foreign makers use this 
system for their high-grade lenses, but still cling to 
arbitrary letters or numbers for their ordinary output. 

Objectives are of two classes — achromatic and apo- 
chromatic. Those in general use are of the achromatic 
type, and they fulfil all requirements for ordinary work. 
Apochromatic objectives are more highly corrected for 
chromatic and spheric aberration, and represent the 
highest type of microscope lenses produced. They are 
very desirable for photomicrographic and research work, 
but for routine laboratory work do not offer advantages 
commensurate with their great cost. They require the 
use of special " compensating " eye-pieces. 

The " working distance " of an objective should not 
be confused with its focal distance. The former term 
refers to the distance between the front lens of the ob- 
jective, when it is in focus, and the cover-glass. It is 



24 INTRODUCTION 

always less than the focal distance, since the " focal 
point " lies somewhere within the objective; and it 
varies considerably with different makes. Long working 
distance is a very desirable feature. 

Objectives are " corrected " for use under certain 
fixed conditions, and they will give the best results only 
when used under the conditions for which corrected. The 
most important corrections are: (a) For tube-length; 
(b) for thickness of cover-glass; and (c) for the medium 
between objective and cover-glass. 

{a) The tube-length with which an objective is to be 
used is usually engraved upon it — in most cases it is 
i6o mm. The draw-tube of the microscope should be 
pulled out until the proper length is obtained, as indi- 
cated by the graduations on its side. When a nose-piece 
is used, it adds about 15 mm. to the tube-length, and the 
draw-tube must be pushed in for that distance. 

(b) The average No. 2 cover-glass is about the thick- 
ness for which most objectives are corrected — usually 0.17 
or 0.18 mm. Very low powers and oil-immersion objec- 
tives do not require any cover-glass. A cover should 
always be used with high dry lenses, but its exact thick- 
ness is more important in theory than in practice. 
Many immersion objectives have such short working 
distance that only very thin covers can be used. 

(c) The correction for the medium between objective 
and cover-glass is very important. This medium may be 
either air or sorrie fluid, and the objective is hence either 
a " dry " or an " immersion " objective. The immersion 
fluid generally used is cedar oil, which gives great optical 
advantages because its index of refraction is the same as 
that of crown glass. It is obvious that only objectives 



USE OF THE MICROSCOPE 25 

with very short working distance, as the 2 mm., can be 
used with an immersion fluid. 

To use an oil-immersion objective a drop of the cedar 
oil which is prepared for the purpose should be placed 
upon the cover, and the objective lowered into it and 
then brought to a focus in the usual way. 

Bubbles in the oil are a frequent source of trouble, 
and should always be looked for when an immersion 
objective does poor work. They are readily seen by 
removing the eye-piece and looking down the tube. 
Immediately after use the oil should be removed with 
lens-paper or a soft linen handkerchief. 

A useful " pointer " can be made by placing a straight 
piece of a hair across the opening of the diaphragm of 
the eye-piece, cementing one end with a tiny drop of 
balsam, and cutting the hair in two in the middle. When 
the eye-piece is in place, the hair appears as a black Une 
extending from the periphery to the center of the micro- 
scopic field. 

Numeric Aperture. — This expression, usually written 
N. A., indicates the amount of Ught which enters an ob- 
jective from a point in the microscopic field. In optical 
language, N. A. is the sine of one-half the angle of aper- 
ture multiplied by the index of refraction of the medium 
between the cover and the front lens. Numeric aper- 
ture is extremely important, because upon it depends 
resolving power, which is the most important property 
of an objective.' 

* Resolving power really depends upon two factors, the N. A. and the 
wave length of light, but the latter can be ignored in practice. The 
great resolving power of the ultra-microscope depends upon its use of 
light of short wave length. 



26 INTRODUCTION 

Resolving power is the ability to separate minute 
details of structure. For example, the dark portions of 
a good half-tone picture appear gray or black to the un- 
aided eye, but a lens easily resolves this apparently 
uniform surface into a series of separate dots. Resolv- 
ing power does not depend upon magnification. The 
fine lines and dots upon certain diatoms may be brought 
out clearly and crisply {i. e., they are resolved) by an 
objective of high numeric aperture, whereas with an 
objective of lower numeric aperture, but greater magnify- 
ing power, the same diatom may appear to have a smooth 
surface, W'ith no markings at all, no matter how greatly 
it is magnified. Knowing the N. A., it is possible to 
calculate how closely lines and dots may lie and still 
be resolved by a given objective. To state the numeric 
aperture, therefore, is to tell what the objective can 
accomplish; provided, of course, that spheric and chro- 
matic aberrations are satisfactorily corrected. An ob- 
jective's N. A. is usually engraved upon the mounting. 

It is an important fact, and one almost universally 
overlooked by practical microscopists, that the pro- 
portion of the numeric aperture of an objective which is 
utilized depends upon the aperture of the cone of light 
delivered by the condenser. In practice, the numeric 
aperture of an objective is reduced nearly to that of 
the condenser (which is indicated by lower-case letters, 
n. a.).' The condenser should, therefore, have a 
numeric aperture at least equal to that of the objective 
with which it is to be used. Lowering the condenser 

' The N. A. of the objective is not reduced wholly to that of the con- 
denser, because, owing to difraction phenomena, a small part of the un- 
illuminated portion of the black lens is utilized. 



USE OF THE MICROSCOPE 27 

below its focal distance and closing the diaphragm be- 
neath it have the eflfect of reducing its working aperture. 
A condenser, whatever its numeric aperture, cannot 
deliver through the air a cone of light of greater n. a. 
than I. It follows, therefore, that the proper adjust- 
ment of the substage condenser is a matter of great im- 
portance when using objectives of high N. A., and that, 
to gain the full benefit of the resolving power of such 
objectives, the condenser must be focused on the object 
under examination, it must be oiled to the under surface 
of the slide in the same way as the immersion objective 
is oiled to the cover-glass, and the substage diaphragm 
must be wide open. The last condition introduces 
a difficulty in that colorless structures will appear 
"fogged" in a glare of light (Fig. 4). Wright suggests 
that the size of the light source be so regulated by a 
diaphragm that its image, thrown on the slide by the 
condenser, coincides with the real field of the objective, 
and maintains that in this way it is possible to reduce 
the glare of light and to dispel the fog without closing 
the diaphragm of the condenser. 

One can easily determine how much of the aperture 
of an objective is in use by removing the eye-piece, look- 
ing down the tube, and observing what proportion of the 
back lens of the objective is illuminated. The relation 
of the illuminated central portion to the unilluminated 
peripheral zone indicates the proportion of the numeric 
aperture in use. The effect of raising and lowering the 
condenser and of oiling it to the slide can thus be easily 
seen. 

Magnification. — The degree of magnification should 
always be expressed in diameters, not times, which is a 



28 INTRODUCTION 

misleading term. The former refers to increase of 
diameter; the latter, to increase of area. The compara- 
tively low magnification of loo diameters is the same as 
the apparently enormous magnification of 10,000 times. 
The magnifying power of a lens is obtained by dividing 
250 mm., or 10 inches (the distance of normal vision), 
by the focal length of the lens. The focal length of an 
objective is approximately twice the diameter of the 
front lens. Thus, the 2 mm. objective gives a mag- 
nification of 125 diameters; the 25 mm. eye-piece gives 
a magnification of 10 diameters, and is usually designated 
as a 10 X eye-piece. When an objective and eye-piece 
are used together, the total magnification is the product 
of the two. In the case just cited the total magnifica- 
tion would be 1250 diameters. In practice, magnifi- 
cation can be increased in one of three ways: 

(a) Drawing out the tube. Since the increased tube- 
length interferes with spheric correction, it should be 
used only with the knowledge that an imperfect image 
will result. 

(b) Using a higher power objective. As a rule, this is 
the best way, because resolving power is also increased; 
but it is often undesirable because of the shorter working 
distance, and because the higher objective often gives 
greater magnification than is desired, or cuts down the 
size of the real field to too great an extent. 

(c) Using a shorter eye-piece. This is the simplest 
method. It has, however, certain limitations. When 
too high an eye-piece is used, there results a hazy image 
in which no structural detail is seen clearly. This is 
called " empty magnification," and depends upon the 
fact that the objective has not sufl[icient resolving power 



USE OF THE MICROSCOPE 29 

to support the high magnification. The extent to which 
magnification can be satisfactorily increased by eye- 
piecing depends wholly upon the resolving power of the 
objective, and consequently upon the N. A. The great- 
est total or combined magnification which will give an 
absolutely crisp picture is found by multiplying the N. A. 
of an objective by 400. The greatest magnification 
which can be used at all satisfactorily is 1000 times the 
N. A. For example: The ordinary 2 mm. objective has 
a N. A. of 1.30; the greatest magnification which will 
give an absolutely sharp picture is 520 diameters^ which 
is obtained approximately by using a 4X eye-piece. 
Higher eye-pieces can be used, up to a total magnifica- 
tion of 1300 diameters (10 X eye-piece), beyond which 
the image becomes wholly unsatisfactory. 

Focusing. — It is always best to " focus up," which 
saves annoyance and probable damage to slides and ob- 
jectives. This is accomplished by bringing the objec- 
tive nearer the slide than the proper focus, and then, with 
the eye at the eye-piece, turning the tube up tmtil the 
object is clearly seen. The fine adjustment should he 
used only to get an exact focus with the higher power ob- 
jectives after the instrument is in approximate focus. 
It should not be turned more than one revolution. 

There will be less fatigue to the eyes if both are kept 
open while using the microscope, and if no effort is made 
to see objects which are out of distinct focus. Fine 
focusing should be done with the fine adjustment, not 
with the eye. An experienced microscopist keeps his 
fingers almost constantly upon one or other of the focus- 
ing adjustments. Greater skill in recognizing objects 
will be acquired if the same eye be always used. To be 



3© INTRODUCTION 

seen most clearly, an object should be brought to the 
center of the field. 

Care of the Microscope. — The microscope is a deH- 
cate instrument and should be handled accordingly. It 
is so heavy that one is apt to forget that parts of it are 
fragile. It seems unnecessary to say that when there 
is unusual resistance to any manipulation, force should 
never be used to overcome it until its cause has first 
been sought; and yet it is no uncommon thing to see 
students, and even graduates, push a high-power objec- 
tive against a microscopic preparation with such force 
as to break not only the cover-glass, but even a heavy 
slide. 

It is most convenient to carry a microscope with the 
fingers grasping the pillar and the arm which holds the 
tube; but since this throws a strain upon the fine adjust- 
ment, it is safer to carry it by the base. In the more 
recent instruments a convenient handle-arm is provided. 
To bend the instrument at the joint, the force should 
be applied to the pillar and never to the tube or the stage. 

Lens surfaces which have been exposed to dust only 
should be cleaned with a camel's-hair brush. Those 
which are exposed to finger-marks should be cleaned with 
lens paper, or a soft linen handkerchief wet with saliva. 
Particles of dirt which are seen in the field are upon the 
slide, the eye-piece, or the condenser. Their location 
can be determined by moving the slide, rotating the eye- 
piece, and lowering the condenser. When the image is 
hazy, the objective probably needs cleaning; or in case of 
an oil-immersion lens, there may be bubbles in the oil. 

Oil and balsam which have dried upon the lenses and 
resist saliva may be removed with alcohol or xylol; but 



USE OF THE MICROSCOPE 3! 

these solvents must be used sparingly and carefully, as 
there is danger of softening the cement. Care must be 
taken not to get any alcohol upon the brass parts, as it 
will remove the lacquer. Balsam and dried oil are best 
removed from the brass parts with xylol. 

Choice of a Microscope. — It is poor economy to buy 
a cheap instrument. 

For the work of a clinical laboratory the microscope 
should preferably be of the new handle-arm type, and 
should have a large stage. It should be provided with a 
substage condenser (preferably of 1.40 n. a.), three or 
more objectives, and two or more eye-pieces. 

The most generally useful objectives are: 16 mm., 
4 mm., and 2 mm. oil immersion. The 4 mm. objective 
may be obtained with N. A. of 0.65 or 0.85. If it is to 
be used for blood-counting, the former is preferable, since 
its working distance is sufficient to take the thick cover 
of the Thoma-Zeiss instrument. For coarse objects a 
32 mm. objective is very desirable. The eye-pieces 
most frequently used are 4 X and 8 X . A very low power 
(2X) and a very high (18 X) will sometimes be found 
useful. The micrometer eye-piece is almost a necessity. 
A mechanical stage, preferably of the attachable type, 
is almost indispensable for blood and certain other work. 

A first-class microscope, of either American or foreign 
make, equipped as just described, will cost in the neigh- 
borhood of a hundred dollars, exclusive of the mechanical 
stage. 

- Measurement of Microscopic Objects. — Of the several 
methods, the most convenient is the use of a micrometer 
eye-piece. In its simplest form this is similar to an 
ordinary eye-piece, but has within it a glass disc upon 



32 INTRODUCTION 

which is ruled a graduated scale. When this eye-piece is 
placed in the tube of the microscope, the ruled lines ap- 
pear in the microscopic field, and the size of an object is 
readily determined in terms of the divisions of this scale. 
The value of these divisions in inches or millimeters 
manifestly varies with different magnifications. Their 
value must, therefore, be determined separately for each 
objective. This is accomplished through use of a stage 
micrometer — a glass slide with carefully ruled scale 
divided into hundredths and thousandths of an inch, or 
into subdivisions of a millimeter. • The stage micrometer 
is placed upon the stage of the microscope and brought 
into focus. From the number of divisions of the eye- 
piece scale corresponding to each division of the stage 
micrometer the value of the former in fractions of an 
inch or millimeter is easily calculated. The counting 
slide of the Thoma-Zeiss hemocytometer will answer 
in place of a stage micrometer, the lines which form the 
sides of the small squares being one-twentieth of a milli- 
meter apart. Any eye-piece can be converted into a 
micrometer eye-piece by placing a micrometer disc — 
a small circular glass plate with ruled scale — ruled side 
down upon its diaphragm. 

The principal microscopic objects which are measured 
clinically are animal parasites and their ova and abnor- 
mal blood-corpuscles. The metric system is used almost 
exclusively. For very small objects o.ooi mm. has been 
adopted as the unit of measurement, under the name 
micron. It is represented by the Greek letter y.. For 
larger objects, where exact measurement is not essential, 
the diameter of a red blood-corpuscle (7 to 8 /u) is some- 
times taken as a unit. 



USE OF THE MICROSCOPE 33 

Tuttle has suggested that in feces and other examina- 
tions a little lycopodium powder be mixed with the 
material. The granules are of uniform size — 30 (i in 
diameter — and are easily recognized (Fig. 5). They 
furnish a useful standard with which the size of other 
structures can be compared. 




Fig. s- — Egg of Tsenia saginata. Lycopodium granules used as micrometer (X 250) 
(photograph by the author). 



Photoniicrography. — Very satisfactory pictures of mi- 
croscopic structures can be made by any one with simple 
apparatus. 

Any camera with focusing screen or a Kodak with 
plate attachment may be used. It is best, but not neces- 
sary, to remove the photographic lens. The camera is 
placed with the lens (or lens-opening, if the lens has been 
removed) looking into the eye-piece of the microscope, 
which may be in either the vertical or the horizontal 
position. One can easily rig up a standard to which the 
camera can be attached in the proper position by means 
of a tripod screw. A light-tight connection can be made 
3 



34 INTRODUCTION 

of a cylinder of paper or a cloth sleeve with draw-strings. 
The image will be thrown upon the ground-glass focusing 
screen, and is focused by means of the fine adjustment of 
the microscope. The degree of magnification is ascer- 
tained by placing the ruled plate of the blood-counting 
instrument upon the microscope and measuring the 
image on the screen. The desired magnification is 
obtained by changing objectives or eye-pieces or length- 
ening the camera-draw. 

Focusing is comparatively easy with low powers, but 
when using an oil-immersion objective, it is a difficult 
problem unless the source of light be very brilliant. If 
one always uses the same length of camera and micro- 
scope tube, a good plan is as follows: Ascertain by trial 
with a strong light how far the fine adjustment screw 
must be turned from the correct eye focus to bring the 
image into sharp focus upon the ground-glass screen. 
At any future time one has only to focus accurately 
with the eye, bring the camera into position, and turn the 
fine adjustment the required distance to right or left. 

The Hght should be as intense as possible, in order to 
shorten exposure, but any light that is satisfactory for 
ordinary microscopic work will answer. It is nearly 
always necessary to insert a color screen between the 
light and the microscope. Pieces of colored window- 
glass are useful for this purpose. The screen should have 
a color complementary to that which it is desired to bring 
out strongly in the photograph: for blue structures, a 
yellow screen; for red structures, a green screen. For 
the average stained preparation, a picric-acid yellow or a 
yellow green will be found satisfactory. 

Very fair pictures can be made on Kodak film, but 



USE OF THE MICROSCOPE 35 

orthochromatic plates (of which Cramer's " Iso " and 
Seed's " Ortho " are examples) give much better re- 
sults. The length of exposure depends upon so many 
factors that it can be determined only by trial. It 
will probably vary from a few seconds to fifteen minutes. 
Plates are developed in the usual way,— the tank method 
yielding most uniform and satisfactory results, — but in 
order to secure all the contrast possible, they should be 
considerably overdeveloped. 




Fig. 6. — ^Leukemic blood (about X 650). Photograph taken with a iiodak, as described 

in the text. 



The photograph from which Fig. 6 was made was taken 
with a Kodak and plate attachment on an " Iso " plate, 
the source of light being the electric lamp and condensing 
lens illustrated in Fig. 2. It was focused by the method 
described above. The screen was a picric-acid stained 
photographic plate. Exposure, three and a half min- 
utes. The picture loses considerable detail in reproduc- 
tion. 



CHAPTER I 

THE SPUTUM 

Preliminary Considerations. — Before beginning the 
study of the sputum, the student will do well to familiar- 
ize himself with the structures which may be present 
in the normal mouth, and which frequently appear in the 
sputum as contaminations. Nasal mucus and material 
obtained by scraping the tongue and about the teeth 
should be studied as described for unstained sputum. A 
drop of Lugol's solution should then be placed at the 
edge of the cover, and, as it runs under, the effect upon 
different structures noted. Another portion should be 
spread upon slides or covers and stained by some simple 
stain and by Gram's method. The structures likely to 
be encountered are epithelial cells of columnar and 
squamous types, leukocytes, food-particles, Lepto- 
thrix huccalis, and great numbers of saprophytic bac- 
teria, frequently including spirochetes. These struc- 
tures are described later. 

The morning sputum or the whole amount for twenty- 
four hours should be collected for examination. In 
beginning tuberculosis tubercle bacilli can often be found 
in that first coughed up in the morning when they can- 
not be detected at any other time of day. Sometimes, in 
these early cases, there are only a few mucopurulent flakes 
which contain the bacilH, or only a small purulent mass 
every few days, and these may easily be overlooked. 

36 



THE SPUTUM 37 

Patients should be instructed to rinse the mouth 
well in order to avoid contamination with food-particles 
which may prove confusing in the examination, and to 
make sure that the sputum comes from the lungs or 
bronchi and not from the nose and nasopharynx. Many 
persons find it difficult to distinguish between the two. 
It is always desirable that the material be raised with a 
distinct expulsive cough, but this is not always possible. 
Material from the upper air-passages can usually be iden- 
tified from the large proportion of mucus and the charac- 
ter of the epithelial cells. 

As a receptacle for the sputum, a clean, wide-mouthed 
bottle with tightly fitting cork may be used. The pa- 
tient must be particularly cautioned against smearing 
any of it upon the outside of the bottle. This is prob- 
ably the chief source of danger to those who examine 
sputum. Disinfectants should not be added. They 
so alter the character of the sputum as to render it 
unfit for satisfactory examination. 

"^Tien the examination is begun, the material should 
be spread out in a thin layer in a Petri dish, or between 
two small plates of glass, Hke photographic plates. 
It may then be examined with the naked eye — best 
over a black background — or with a low power of the 
microscope. The portions most suitable for further 
examination may thus be easily selected. This macro- 
scopic examination should never he omitted. 

After an examination the sputum must be destroyed 
by heat or chemicals, and everything which has come in 
contact with it must be sterilized . The utmost care must 
be taken not to allow any of it to dry and become dis- 
seminated through the air. It is a good plan to con- 



38 THE SPUTUM 

duct the examination upon a large newspaper, which 
can then be burned. Contamination of the work table 
is thus avoided. If this is not feasible, the table should 
be washed off with 10 per cent, lysol solution, and allowed 
to dry slowly, as soon as the sputum work is finished. 

Examination of the sputum is most conveniently con- 
sidered under four heads: I. Physical examination. 
II. Microscopic examination. III. Chemic examination. 
IV. Characteristics of the sputum in various diseases. 

I. PHYSICAL EXAMINATION 

1. Quantity. — The quantity expectorated in twenty- 
four hours varies greatly. It may be so slight as to be 
overlooked entirely in beginning tuberculosis. It is 
usually small in acute bronchitis and lobar pneumonia. 
It may be very large — sometimes as much as 1000 c.c. — 
in advanced tuberculosis with large cavities, edema of the 
lung, bronchiectasis, and following rupture of an abscess 
or empyema. It is desirable to obtain a general idea of 
the quantity, but accurate measurement is unnecessary. 

2. Color. — Since the sputum ordinarily consists of 
varying proportions of mucus and pus, it may vary from 
a colorless, translucent mucus to an opaque, whitish or 
yellow, purulent mass. A yellowish green is frequently 
seen in advanced phthisis and chronic bronchitis. In 
jaundice, in caseous pneumonia, and in slowly resolv- 
ing lobar pneumonia it may assume a bright green color, 
due to bile or altered blood-pigment. 

A red color usually indicates the presence of blood. 
Bright red blood, most commonly in streaks, is strongly 
suggestive of phthisis. It may be noted very early in the 
disease. A rusty red sputum is the rule in croupous 



PHYSICAL EXAMINATION 39 

pneumonia, and was at one time considered pathogno- 
monic of the disease. " Prune- juice " sputum is said to 
be characteristic of '' drunkard's pneumonia." It at 
least indicates a dangerous type of the disease. A 
brown color, due to altered blood-pigment, follows 
hemorrhages from the lungs, and is present, to greater or 
less degree, in chronic passive congestion of the lung, 
which is most frequently due to a heart lesion. 

Gray or black sputum is observed among those who 
work much in coal-dust, and is occasionally seen in 
smokers who are accustomed to " inhale." 

3. Consistence. — According to their consistence, sputa 
are usually classified as serous, mucoid, purulent, sero- 
purulent, mucopurulent, etc., which names explain them- 
selves. As a rule, the more mucus and the less pus and 
serum a sputum contains, the more tenacious it is. 

The rusty sputum of croupous pneumonia is extremely 
tenacious, so that the vessel in which it is contained may 
be inverted without spilling it. The same is true of the 
almost purely mucoid sputum (" sputum crudum ") of 
beginning acute bronchitis, and of that which follows 
an attack of asthma. A purely serous sputum, usually 
slightly blood tinged, is fairly characteristic of edema of 
the lungs. 

4. Dittrich's Plugs. — While these bodies sometimes 
appear in the sputum, they are more frequently ex- 
pectorated alone. They are caseous masses, usually 
about the size of a pin-head, but sometimes reaching 
that of a bean. The smaller ones are yellow, the larger 
ones gray. When crushed, they emit a foul odor. 
Microscopically, they consist of granular debris, fat-glob- 
ules, fatty acid crystals, and bacteria. They are formed 



40 THE SPUTUM 

in the bronchi, and are sometimes expectorated by 
healthy persons, but are more frequent in putrid bron- 
chitis and bronchiectasis. The laity commonly regard 
them as evidence of tuberculosis. The similar caseous 
masses which are formed in the crj'pts of the tonsils are 
sometimes also included under this name. 

11. MICROSCOPIC EXAMINATION 

The portions most likely to contain structures of 
interest should be very carefully selected, as already 
described. The few minutes spent in this preliminary 
examination will sometimes save hours of work later. 
Opaque, white or yellow particles are most frequently 
bits of food, but may be cheesy masses from the tonsils; 
small cheesy nodules, derived from tuberculous cavities 
and containing many tubercle bacilli and elastic fibers; 
Curschmann's spirals, or small fibrinous casts, coiled into 
little balls; or shreds of mucus with great numbers of 
entangled pus-corpuscles. The food-particles most apt 
to cause confusion are bits of bread, which can be recog- 
nized by the blue color which they assume when touched 
with iodin solution. 

Some structures are best identified without staining; 
others require that the sputum be stained. 

A. Unstained Sputum 

A careful study of the unstained sputum should be 
included in every routine examination. It best reveals 
certain structures which are seen imperfectly or not at 
all in stained preparations. It gives a general idea of 
the other structures which are present, such as pus- 



MICROSCOPIC EXAMINATION 41 

corpu§cles, eosinophiles, epithelial cells, and blood, and 
thus suggests appropriate stains to be used later. 

The particle selected for examination should be trans- 
ferred to a clean sUde, covered with a clean cover-glass, 
and examined with the i6 mm. objective, followed by 
the 4 mm. It is convenient to handle the bits of 
sputum with a wooden tooth-pick or with a wooden 
cotton-applicator, which may be burned when done with. 
The platinum wire used in bacteriologic work is less 
satisfactory because not usually stiff enough. 

The more important structures to be seen in unstained 
sputum are: elastic fibers, Curschmann's spirals, Char- 
cot-Leyden crystals, fibrinous casts, the ray fungus of 
actinomycosis, and molds. Pigmented cells, especially 
the so-called " heart-failure cells " (p. 62), are also best 
studied without staining (Plate II, Fig. i). 

1. Elastic Fibers.— These are the elastic fibers of 
the pulmonary substance (Fig. 7). When found in the 
sputum, they always indicate destructive disease of the 
lung, provided they do not come from the food, which is 
a not infrequent source. They are found most com- 
monly in phthisis; rarely in other diseases. Advanced 
cases of tuberculosis often show great numbers, and, 
rarely, they may be found in early tuberculosis when the 
bacilli cannot be detected. In gangrene of the lung, 
contrary to the older teaching, elastic tissue is probably 
always present in the sputum, usually in large fragments. 

The fibers should be searched for with a 16 mm. 
objective, although a higher power is needed to identify 
them with certainty. Under the 4 mm. they appear as 
slender, highly refractive fibers with double contour, and 
often curled or spHt ends. Frequently they are found 



42 THE SPUTUM 

in alveolar arrangement, retaining the original outline of 
the alveoli of the lung (Fig. 7, 6). This arrangement 
is positive proof of their origin in the lung. Leptothrix 
buccalis, which is a normal inhabitant of the mouth, 
may easily be mistaken for elastic tissue. It can be dis- 
tinguished by running a little iodin solution under the 
cover-glass (see p. 56). 




Fig. 7. — Elastic fibers from the sputum: o, Highly magnified; b, alveolar arrangement, 
less highly magnified (after Bizzozero). 



Fatty-acid crystals, which are often present in Dit- 
trich's plugs and in sputum which has lain in the body 
for some time, also simulate elastic tissue when very 
long, but they are more like stiff, straight or curved 
needles than wavy threads. They show varicosities 
when the cover-glass is pressed upon. The structures 
which most frequently confuse the student are the cotton 
fibrils which are present as a contamination in most 



MICROSCOPIC EXAMINATION 43 

sputa. These are usually coarser than elastic fibers, and 
flat, with one or two twists, and often have longitudinal 
striations and frayed-out ends. 

To find elastic fibers when not abundant, boil the 
sputum with a 10 per cent, solution of caustic soda until 
it becomes fluid; add several times its bulk of water, and 
centrifugalize, or allow to stand for twenty-four hours in 
a conical glass. Examine the sediment microscopically. 
The fibers will be pale and swollen and, therefore, 
somewhat difficult to recognize. Too long boiling will 
destroy them entirely. 

The above procedure, although widely recommended, 
will rarely or never be necessary if the sputum is care- 
fully examined in a thin layer against a black back- 
ground macroscopically and with a hand-lens, and if all 
suspicious portions are further studied with the micro- 
scope. 

2. Curschmann's Spirals.— These pecuHar structures 
are found most frequently in bronchial asthma, of 
which they are fairly characteristic. They may occa- 
sionally be met with in chronic bronchitis and other 
conditions. Their nature has not been definitely deter- 
mined. 

Macroscopically, they are whitish or yellow, twisted 
threads, frequently coiled into little balls (Fig. 8, I). 
Their length is rarely over half an inch, though it some- 
times exceeds two inches. Under a 16 mm. objective 
they appear as mucous threads having a clear central 
fiber, about which are wound many fine fibrils (Fig. 8, 
II. and III.). Eosinophiles are usually present within 
them, and sometimes Charcot-Leyden crystals. Not 
infrequently the spirals are imperfectly formed, con- 



44 



THE SPUTUM 



sisting merely of twisted strands of mucus inclosing 
leukocytes. The central fiber is absent from these. 

3. Charcot=Leyden Crystals.— Of the crystals which 
may be found in the sputum, the most interesting are the 
Charcot-Leyden crystals. They may be absent when 
the sputum is expectorated, and appear in large numbers 
after it has stood for some time. They are rarely found 




,M 



I. II III. 

Fig. 8. — Curschmann's spirals: I., Natural size; II. and HI., enlarged: a, central fiber 
(after Curschmann). 



except in cases of bronchial asthma, and were at one 
time thought to be the cause of the disease. They 
frequently adhere to Curschmann spirals. Their exact 
nature is unknown. Their formation seems to be in 
some way connected with the presence of eosinophilic 
cells. Outside of the sputum they are found in the 
feces in association with animal parasites, and in the 
coagulated blood in leukemia. 



MICROSCOPIC EXAMINATION 



45 



They are colorless, pointed, often needle-like, octa- 
hedral crystals (Fig. 9). Their size varies greatly, the 
average length being about three or four times the 
diameter of a red blood-corpuscle. 




Fig. 9. — Charcot-Leyden crystals (after Riegel). 

Other crystals — hematoidin, cholesterin, and, most 
frequently, fatty-acid needles (see Fig. 36)^ — are common 
in sputum which has remained in the body for a consider- 
able time, as in abscess of the lung and bronchiectasis. 

4. Fibrinous Casts.— These are casts of the bronchi, 
frequently, but not always, composed of fibrin. In 
color they are usually white or grayish, but may be 
reddish or brown, from the presence of blood-pigment. 
Their size varies with that of the bronchi in which they 
are formed. They may, rarely, be fifteen or more 
centimeters in length. When large, they can be recog- 



46 THE SPUTUM 

nized with the naked eye by floating them out in water 
over a black surface; when small, a low power of the 
microscope must be used. Their branching, tree-like 
structure (Fig. 10) is usuall}^ sufficient to identify them. 
Fibrinous casts are characteristic of fibrinous bron- 
chitis, but may also be found in diphtheria of the smaller 
bronchi. Very small casts are often seen in croupous 
pneumonia. 




Fig. 10. — Fibrinous bronchial cast (Sahli). 

5. Actinomyces Bovis (Ray=fungus). — In the sputum 
of pulmonary actinomycosis and in the pus from actino- 
mycotic lesions elsewhere small, yellowish, " sulphur " 
granules can be detected with the unaided eye. With- 
out a careful macroscopic examination they are almost 
certain to be overlooked. The fungus can be seen by 
crushing one of these granules between slide and cover, 
and examining with a low power. It consists of a net- 



MICROSCOPIC EXAMINATION 



47 



work of threads having a more or less radial arrangement, 
those at the periphery presenting club-shaped extremi- 
ties (Fig. ii). It can be brought out more clearly by 
running a little solution of eosin in alcohol and glycerin 
under the cover. This organism, also called Strepto- 
thrix actinomyces, apparently stands midway between 
the bacteria and the molds. It stains by Gram's method. 
Actinomycosis of the lung is rare. The clinical pic- 
ture is that of tuberculosis. 




Fig. n. — Sputum from a case of actinomycosis; stained (Jakob). 

6. Molds and Yeasts.— The hyphae and spores of 
various molds are occasionally met with in the sputum. 
They are usually the result of contamination, and have 
little significance. The hyphae are rods, usually jointed 
or branched (Fig. 62), and often arranged in a mesh work 
(myceHum); the spores are highly refractive spheres. 
Both stain well with the ordinary stains. 

In the extremely rare condition of systemic blasto- 
mycosis the specific yeasts have been found in the sputum 



48 THE SPUTUM 

in large numbers. It is advisable to add a little lo per 
cent, caustic soda solution and examine unstained. 

7. Animal Parasites.— These are extremely rare in 
the sputum in this country. A trichomonad, perhaps 
identical with Trichomonas vaginalis, has been seen in 
the sputum of putrid bronchitis and gangrene of the 
lung, but its causal relationship is doubtful. In Japan, 
infection with the lung flukeworm, Paragonimus wes- 
termani, is common, and the ova are found in the 
sputum. The lung is not an uncommon seat for echino- 
coccus cysts, and booklets and scolices may appear, as 
may also A moeba histolytica, when a hepatic abscess has 
ruptured into the lung. Ciliated body-cells, with cilia 
in active motion, are not infrequently seen, and may 
easily be mistaken for infusoria. All the above-men- 
tioned parasites are described in Chapter VI. 

B. Stained Sputum 

Structures which are best seen in stained sputum are 
bacteria and cells. 

A number of smears should be made upon slides or 
covers, dried in the air, and fixed in the flame, as de- 
scribed on the next page. Fixation will kill the bac- 
teria when covers are used, and the smears may be kept 
indefinitely; but smears on slides are often not sterile, 
and should be handled accordingly. One of the smears 
should be stained with some simple stain, like Lofiler's 
methylene-blue, which will give a good idea of the 
various cells and bacteria present. Special stains may 
then be applied, as indicated, but a routine examination 
should, in all cases, include a stain by the method for 
the tubercle bacillus and by Gram's method. 



MICROSCOPIC EXAMINATION 49 

1 . Bacteria. — Saprophytic bacteria from mouth con- 
tamination are frequently present in large numbers and 
will prove confusing to the inexperienced. The pres- 
ence of squamous cells in their neighborhood will sug- 
gest their source. Among the pathogenic organisms 
which have clinical importance are: tubercle bacilli; 
staphylococci and streptococci; pneumococci; bacilli of 
Friedlander; influenza bacilU, and Micrococcus catar- 
rhdlis. 

(i) Tubercle Bacillus. — The presence of the tubercle 
bacillus may be taken as positive evidence of the ex- 
istence of tuberculosis somewhere along the respiratory 
tract, most likely in the lung. In laryngeal tuberculosis 
it is not easily found in the sputum, but can fre- 
quently be detected in swabs made directly from the 
larynx. 

Recognition of the tubercle bacillus depends upon the 
fact that it stains with difficulty; but that when once 
stained, it retains the stain tenaciously, even when 
treated with a mineral acid, which quickly removes the 
stain from other bacteria. This " acid-fast " property 
is due to the presence of a waxy capsule. The most 
convenient method for general purposes is here given 
in detail: 

Gabbet's Method. — (i) Spread suspicious particles thinly 
and evenly upon a slide or a cover-glass held in the grasp of 
cover-glass forceps. In general, slides are more satisfactory, 
but cover-glasses are easier to handle while staining. Do not 
grasp a cover too near the edge or the stain will not stay 
on it well. Tenacious sputum will spread better if gently 
warmed while spreading. 

(2) Dry the film in the air. 
4 



50 THE SPUTUM 

(3) Fix in a flame; i. e., pass the cover-glass rather slowly, 
with film side up, three times (a slide about twelve times) 
through the flame of a Bunsen burner or alcohol lamp low 
down in the flame. Take care not to scorch. Should the 
film be washed off during future manipulations, fixation has 
been insufficient. 

(4) Apply as much carbolfuchsin as will stay on, and hold 
over a flame so that it will steam for three minutes or longer, 
replacing the stain as it evaporates. If the bacilli are well 
stained in this step, there will be little danger of decolorizing 
them later. Too great heat will interfere with the staining 
of some of the bacilli, probably by destroying the waxy 
envelop upon which the acid-fast property depends. It is 
better to stain at room temperature for twelve to twenty- 
four hours. 

(5) Wash the film in water. 

(6) Apply Gabbet's stain to the under side of the cover- 
glass to remove excess of carbolfuchsin, and then to the film 
side. Allow this to act for one-fourth to one-half minute. 

(7) Wash in water. 

(8) If, now, the thinner portions of the film are blue, pro- 
ceed to the next step; if they are still red, repeat steps (6) 
and (7) until the red has disappeared. Too long application 
of Gabbet's stain will decolorize the tubercle bacilli. 

(9) Place the preparation between layers of filter-paper and 
dry by rubbing with the fingers, as one would in blotting ink, 

(10) Put a drop of Canada balsam upon a clean slide, place 
the cover-glass film side down upon it, and examine with an 
immersion objective. Cedar oil or water may be used in 
place of balsam for temporary preparations. Smears on 
slides may be examined directly with an oil-immersion lens, 
no cover being necessary. 

Carbolfuchsin is prepared by mixing 10 c.c. of a saturated 
alcoholic solution of fuchsin with 90 c.c. of 5 per cent, aqueous 
solution of phenol. 



MICROSCOPIC EXAMINATLON CI 

Gabbet's stain consists of n^^^ylene-bIue/0 2 §nt\,;^25 per 
cent, sulphuric acid, loo c.c. '^^^-l/> fir ^ ' ^^^f^fiTrj 

Both stains can be purchased ready prepared; -'^ Up f-_^ 

Other Methods. — The objection is often raised that de- 'v /^ 
colorization is masked by the blue in Gabbet's stain, but this 
will not make trouble if step 8 is carefully carried out. The 
Ziehl-Neelsen method is preferred by many: After the stain- 
ing with carbolfuchsin the smear is washed in 5 per cent, 
nitric acid until decolorized, washed in water, stained lightly 
with LofHer's methylene-blue, again washed, and mounted. 

Pappenheim's Method. — This is the same as Gabbet's 
method, except that Pappenheim's methylene-blue solution 
is substituted for Gabbet's solution. This consists of: 

Corallin (rosolic acid) i gm. 

Absolute alcohol 100 c.c. 

Saturate with methylene-blue and add 20 c.c. glycerin. 

The method is very satisfactory for routine work. De- 
colorization of the tubercle bacillus is practically impos- 
sible: it retains its red color, even when soaked overnight 
in Pappenheim's solution. The stain was originally recom- 
mended as a means of differentiating the smegma bacillus, 
which is decolorized by it; but it is not to be absolutely relied 
upon for this purpose. 

In films stained by these methods tubercle bacilli, 
if present, will be seen as slender red rods upon a blue 
background of mucus and cells (Plate. II, Fig. 2). They 
average 3 to 4 ll in length — about one-half the diameter 
of a red blood-corpuscle. Beginners must be warned 
against mistaking the edges of cells, or particles which 
have retained the red stain, for bacilli. The appear- 
ance of the bacilli is almost always typical, and if there 



52 THE SPUTUM 

seems room for doubt, the structure in question is prob- 
ably not a tubercJe bacillus. They may lie singly or in 
groujw. They are very frequently bent and often have 
a beaded appearance. It is possible that the larger, 
beaded bacilli indicate a less active tuberculous process 
than do the smaller, uniformly stained ones. Some- 
times they are present in great numbers — thousands 
in a field of the 2 mm. objective. Sometimes sev- 
eral cover-glasses must be examined to find a single 
bacillus. At times they are so few that none are found 
in stained smears, and special methods are required 
to detect them. The number may bear some relation 
to the severity of the disease, but this relation is by no 
means constant. The mucoid sputum from an incip- 
ient case sometimes contains great numbers, while 
sputum from large tuberculous cavities at times contains 
very few. Failure to find them is not conclusive, 
though their absence is much more significant when the 
sputum is purulent than when it is mucoid. 

When they are not found in suspicious cases, one of 
the following methods should be tried: 

(i) Antiformin Method. — This has lately come into use, 
and has superseded the older methods of concentration. 
The chief difficulty with the older methods, such as boiling 
with caustic soda, is that the bacilli are so injured in the 
process that they do not stain characteristically. 

Antiformin is the patented name for a preparation con- 
sisting essentially of equal parts of a 15 per cent, solution of 
caustic soda and a 20 per cent, solution of sodium hypo- 
chlorite. It keeps fairly well. The sputum is thoroughly 
shaken in a corked bottle with one-fourth its volume of anti- 
formin, and allowed to stand four to six hours in an incubator, 



PLATE II 




Fig. I. — Heart-failure cells in sputum, containing blood-pigment, from 
a case of cardiac congestion of the lungs (Jakob). 




Fig. 2. — A, Sputum showing tubercle bacilli stained with car- 
bolfuchsin and Gabbet's methylene-blue solution (obj. one-twelfth 
oil-immersion); B, sputum of anthracosis, showing particles of coal-dust 
stained with methylene-blue (obj. one-twelfth oil-immersion) (Boston). 



MICROSCOPIC EXAMINATION 53 

or twenty-four hours at room-temperature. The sputum will 
be thoroughly liquefied. A centrifuge tubeful is thoroughly 
centrifugalized, the supernatant fluid is poured off and 
replaced with water, and centrifugalization is repeated. 
This washing is repeated several times. Some of the sedi- 
ment is then spread upon slides (with a little egg-albumen 
or some of the untreated sputum to cause it to adhere), 
dried, fixed, and stained. The tubercle bacilli are not killed, 
but retain their form and staining properties unchanged. 
Other bacteria and cells are destroyed. 

Since the bacilli remain alive, the utmost care must be 
used in handling, and all tubes and glassware which have 
come in contact with the liquefied sputum must be 
sterilized. 

(2) Animal Inoculation. — Inoculation of guinea-pigs is 
the court of last appeal in detection of tubercle bacilli. The 
method is described on p. 375-- 

There are a number of bacilli, called acid-fast bacilli, 
which stain in the same way as the tubercle bacillus. 
They stain with difficulty, and when once stained, retain 
the color even when treated with a mineral acid; but, 
unlike the tubercle bacillus, most of them can be decolor- 
ized with alcohol. Of these, the smegma bacillus is the 
only one likely ever to cause confusion. It, or a similar 
bacillus, is sometimes found in the sputum of gangrene 
of the lung. It occurs normally about the glans penis 
and the clitoris, and is often present in the urine and in 
the wax of the ear. The method of distinguishing it 
from" the tubercle bacillus is given later (p. 168). 

Other bacteria than the acid-fast group are stained 
blue by Gabbet's method. Those most commonly found 
are staphylococci, streptococci, and pneumococci. Their 



54 THE SPUTUM 

presence in company with the tubercle bacillus consti- 
tutes mixed infection, which is much more serious than 
single infection by the tubercle bacillus. It is to be 
remembered, however, that a few of these bacteria may 
reach the sputum from the upper air-passages. Clini- 
cally, mixed infection is evidenced by fever. 

(2) Staphylococcus and Streptococcus (p. 368). — One 
or both of these organisms is commonly present in com- 
pany with the tubercle bacillus in the sputum of ad- 
vanced phthisis (Plate II, Fig. 2). They are often 
found in bronchitis, catarrhal pneumonia, and many 
other conditions. 

(3) Pneumococcus (Diplococcus of Frankel). — ^The 
pneumococcus is the causative agent in nearly all cases 
of croupous pneumonia, and is commonly found in large 
numbers in the rusty sputum of this disease. It is some- 
times met with in the sputum of catarrhal pneumonia, 
bronchitis, and tuberculosis. It is also an important 
factor in the causation of pleurisy, meningitis, otitis 
media, and other inflammations. It has been found in 
the saliva in health. Pneumococci are about the size of 
streptococci. They are ovoid in shape, and lie in pairs, 
end to end, often forming short chains. Each is sur- 
rounded by a gelatinous capsule, which is its distinctive 
feature (Fig. 12). Diplococci without capsules are com- 
mon in the sputum, but have no special significance. 

The pneumococcus is closely related to the strepto- 
coccus, and it is sometimes extremely difficult to differ- 
entiate them even by culture methods (for which see 
p. 368). The morphology of the pneumococcus, the 
fact that it is Gram-positive, and the presence of a cap- 
sule are, however, generally sufficient for its recognition 



MICROSCOPIC EXAMINATION 55 

in smears from sputum or pus. The capsule is often 
seen as a halo around pairs of cocci in smears stained 
by the ordinary methods, particularly Gram's method, 
but to show it well special methods are required. 
There are numerous special methods of staining cap- 
sules which are applicable to other encapsulated bacteria, 
as well as to the pneumococcus, but few of them are 
satisfactory. Buerger's method can be recommended. 
It is especially useful with cultures upon serum media, 




Fig. 12. — Diplococcus pneumonix in the blood ( X looo) (Frankel and Pfeiffer). 

but is applicable also to the sputum. Smith's new 
method is easier of application, and apparently gives 
uniformly good results. The India-ink method described 
for the organism of syphilis is likewise said to show 
capsules satisfactorily. The sputum should be fresh — 
not more than three or four hours old. 

Buerger's Method for Capsules. — (i) Mix a few drops 
each of the sputum and blood-serum or egg-albumen solu- 



56 THE SPUTUM 

tion (egg-albumen, distilled water, equal parts; shake and 
filter through cotton). Blood-serum can be obtained as 
described for the Widal test, p. 258. Make thin smears from 
the mixture, and just as the edges begin to dry, cover with 
Miiller's fluid (potassium dichromate, 2.5 gm.; sodium sul- 
phate, i.o gm.; water, 100 c.c.) saturated with mercuric 
chlorid (ordinarily about 5 per cent.). Gently warm over 
a flame for about three seconds. 

(2) Rinse very quickly in water, 

(3) Flush once with alcohol. 

(4) Apply tincture of iodin for one to two minutes. 

(5) Thoroughly wash off the iodin with alcohol and dry in 
the air. 

(6) Stain about three seconds with weak anilin-gentian- 
violet freshly made up as follows: Anilin oil, 10; water, 100; 
shake; filter; and add 5 c.c. of a saturated alcoholic solution 
of gentian violet. 

(7) Rinse off the stain with 2 per cent, solution of sodium 
chlorid, mount in this solution, and examine with a one- 
twelfth objective. 

Buerger suggests a very useful variation as follows: After 
the alcohol wash and drying, the specimen is stained by 
Gram's method (p. 409), counterstained with aqueous solu- 
tion of fuchsin, washed, and mounted in water. The 
pneumococcus holds the purple stain, while all capsules 
take on the pink counterstain. 

Smith's Method. — (i) Make thin smears of the sputum 
or other material, which should be as fresh as possible. 

(2) Fix in the flame in the usual manner. 

(3) Apply a 10 per cent, aqueous solution of phospho- 
molybdic acid (Merck) for four to five seconds. 

(4) Rinse in water. 

(5) Apply anilin-gentian- violet, steaming gently for 
fifteen to thirty seconds. 

(6) Rinse in water. 



MICROSCOPIC EXAMINATION 57 

(7) Apply Gram's iodin solution, steaming gently for 
fifteen to thirty seconds. 

(8) Wash in 95 per cent, alcohol until the purple color 
ceases to come off. 

(9) Rinse in water. 

(10) Apply a 6 per cent, aqueous solution of eosin (Grii- 
bler, w. g.), and gently warm for one-half to one minute. 

(11) Rinse in water. 

(12) Wash in absolute alcohol. 

(13) Clear in xylol. 

(14) Mount in balsam. 

This is essentially Gram's method (seep. 409), preceded 
by treatment with phosphomolybdic acid and followed by 
eosin. Gram-positive bacteria like the pneumococcus are 
deep purple; capsules are pink and stand out clearly. 

When the method is applied to Gram-negative bacteria, 
steps 5 to 9 inclusive are omitted ; between steps 1 1 and 1 2 
the preparation is counterstained with Loffler's methylene- 
blue, gently warming for fifteen to thirty seconds. 

A nilin- gentian-violet. — Ehrlich's formula is the one gener- 
ally used, but this keeps only a few weeks. Stirling's solu- 
tion, which keeps much better and seems to give equal results, 
is as follows: gentian-violet, 5 gm.; alcohol, 10 c.c; anilin 
oil, 2 c.c; water, 88 c.c. 

Formalin- gentian-violet is a satisfactory substitute for 
anilin-gentian-violet and is permanent. It consists of 5 per 
cent, solution formalin, 75 parts; saturated alcohohc solution 
gentian-violet, 25 parts. 

Gram's Iodin Solution. — Iodin, i gm.; potassium iodid, 
2 gm.; water, 300 c.c. 

Loffler^s alkaline methylene-bliie is a very generally useful 
stain for bacteria. It is composed of 30 parts of a saturated 
alcoholic solution of methylene-blue and 100 parts of a 
I : 10,000 aqueous solution of caustic potash. It keeps 
indefinitely. 



58 THE SPUTUM 

(4) Bacillus of Friedlander (Bacillus Mucosus Cap- 
sulatus). — In a small percentage of cases of pneumonia 
this organism is found alone or in company with the 
pneumococcus. Its pathologic significance is uncer- 
tain. It is often present in the respiratory tract under 
normal conditions. Friedliinder's bacilli are non-motile, 
encapsulated rods, sometimes arranged in short chains 
(Fig. 13) . Very short individuals in pairs closely resemble 
pneumococci, from which they are distinguished by the 
fact that they are Gram-decolorizing. 

(5) Bacillus of Influenza. — This 
is the etiologic factor in true in- 
fluenza, although conditions which 
are clinically similar or identical 
may be caused by the pneumococ- 
cus, streptococcus, or Micrococcus 
catarrhalis. It is present, often in 
Friediandcr's large uumbcrs, in the nasal and 

bacillus in pus from pulmon- , , , 

ary abscess (obj. one-twelfth) brouchial secrctions, and is also 
(Boston). found in the local lesions following 

influenza. Chronic infection by influenza bacilli may 
be mistaken clinically for tuberculosis, and they should 
be searched for in all cases of obstinate chronic bron- 
chitis. 

Their recognition depends upon the facts that they 
are extremely small bacilli ; that most of them lie within 
the pus-cells; that their ends stain more deeply than their 
centers, sometimes giving the appearance of minute 
diplococci; and that they are decolorized by Gram's 
method of staining (Figs. 14 and 149). 

They are stained blue in the methods for tubercle 
bacilli, but are more certainly recognized by Gram's 




MICROSCOPIC EXAMINATION 59 

method, followed by a counterstain. Pappenheim's 
pyronin-methyl-green stain is especially satisfactory. 

(6) Micrococcus Catarrhalis. — This organism is fre- 
quently present in the sputum in inflammatory condi- 
tions of the respiratory tract resembling influenza. 
It is sometimes present in the nasal secretions in health. 
It is a Gram-negative diplococcus, frequently intra- 
cellular, and can be distinguished from the meningo- 




Fig. 14. — Bacillus of influenza; cover-glass preparation of sputum from a case of influenza, 
showing the bacilli in leukocytes; highly magnified (Pfeiffer). 



coccus and gonococcus only by means of cultures. It 
grows readily on ordinary media. 

2. Cells. — These include pus-corpuscles, epithelial 
cells, and red blood-corpuscles. 

(i) Pus-corpuscles are present in every sputum, and 
at times the sputum may consist of little else. They are 
the polymorphonuclear leukocytes of the blood, and 
appear as rounded cells with several nuclei or one very 
irregular nucleus (Fig. 11 and Plate II, Fig. 2). They 



6o THE SPUTUM 

are frequently filled with granules of coal-dust and are 
often much degenerated. Such coal-dust-laden leuko- 
cytes are especially abundant in anthracosis, where 
angular black particles, both intra- and extra-cellular, 
are often so numerous as to color the sputum (Plate II, 
Fig. 2, B). Occasionally mononuclear leukocytes are 
present. 

Eosinophilic cells are quite constantly found in large 
numbers in the sputum of bronchial asthma near the 




Fig. 15. — Sputum from a case of asthma showing' Ijukocytes, some containiivg eosino- 
philic granules; free eosinophilic granules and micrococci; stained with eosin and methy- 
lene-blue ( X 350) (Jakob). 

time of the paroxysm, and constitute one of the most 
distinctive features of the sputum of this disease. They 
resemble ordinary pus-corpuscles, except that their 
cytoplasm is filled with coarse granules having a marked 
affinity for eosin. It is worthy of note that many of 
them, sometimes the majority, are mononuclear. Large 
numbers of free granules, derived from disintegrated 
cells, are also found (Fig. 15). 



MICROSCOPIC EXAMINATION 6l 

Ordinary pus-cells are easily recognized in sputum 
stained by any of the methods already given. For 
eosinophilic cells, some method which includes eosin must 
be used. A simple method is to stain the dried and 
fixed film two or three minutes with saturated solution 
of eosin, and then one-half to one minute with Loffler's 
methylene-blue ; nuclei and bacteria will be blue, eosino- 
phiHc granules bright red. 

(2) Epithelial cells may come from any part of the 
respiratory tract. A few are always present, since des- 
quamation of cells goes on constantly. Their recogni- 
tion is important chiefly as an aid in deciding upon the 
source of the portion of the sputum in which they are 
found. In suspected lung conditions it is manifestly 
useless to study material from the nose only, yet this 
is not infrequently done. They have little diagnostic 
value, although a considerable excess would indicate a 
pathologic condition at the site of their origin. Any 
of the stains mentioned above will show them, and they 
can usually be identified in unstained sputum. In 
general, three forms are found: 

(a) Squamous Cells. — Large, flat, polygonal cells with 
a comparatively small nucleus (Fig. 16, i). They come 
from the upper air-passages, and are especially numerous 
in laryngitis and pharyngitis. They are frequently 
studded with bacteria — most commonly diplococci. 

{h) Cylindric Cells from the Nose, Trachea, and Bronchi 
(Fig. 16, /, h). — These are not usually abundant, and, 
as a rule, they are not identified because much altered 
from their original form, being usually round and swollen. 
When very fresh, they may retain their cylindric form, 
sometimes bearing cilia in active motion. 



62 



THE SPUTUM 



(c) Alveolar Cells. — Rather large, round, or oval cells 
with one or two round nuclei (Fig. i6). Their source is 
presumably the pulmonary alveoli. Like the leuko- 
cytes, they frequently contain particles of carbon (nor- 
mal lung pigment). In chronic heart disease, owing to 
long-continued passive congestion, they may be filled 




Fig. i6. — Different morphologic elements of the sputum (unstained): a, b, c. Pulmo- 
nary or alveolar epithelium — a, with normal lung pigment (carbon); 6, with fat-droplets; 
c, with myelin globules; d, pus -corpuscles; e. red blood-corpuscles; /, cylindric beaker- 
shaped bronchial epithelial cells; g, free myelin globules; h. ciliated epithelium of different 
kinds from the nose, altered by coryza; i, squamous cells from the pharynx (after Bizzo- 
zero). 



with brown granules of altered blood-pigment, and are 
then called " heart-failure cells." The presence of 
these cells in considerable numbers, by directing one's 
attention to the heart, will sometimes clear up the 
etiology of a chronic bronchitis. They are best seen in 
unstained sputum, appearing as grayish or colorless 



CHEMIC EXAMINATION 63 

balls filled with rounded granules of brown or yellow 
pigment. (Plate II, Fig. i.) 

Alveolar cells commonly contain fat-droplets and, less 
frequently, myelin globules. The latter are colorless, 
rounded bodies, sometimes resembUng fat-droplets, but 
often showing concentric or irregularly spiral markings 
(Fig. 16, c, g). They are also found free in the sputum. 
They are abundant in the scanty morning sputum of 
apparently healthy persons, but may be present in any 
mucoid sputum. 

(3) Red blood-corpuscles may be present in small 
numbers in almost any sputum. When fairly constantly 
present in considerable numbers, they are suggestive of 
phthisis. The corpuscles, when fresh, are shown by any 
of the staining methods which include eosin. They are 
commonly so much degenerated as to be unrecognizable, 
and often only altered blood-pigment is left. Ordinarily, 
blood in the sputum is sufficiently recognized with the 
naked eye. 

III. CHEMIC EXAMINATION 

There is little to be learned from a chemic examina- 
tion, and it is rarely undertaken. Recently, however, 
it has been shown that the presence or absence of albumin 
may have clinical significance. Albumin is constantly 
present in the sputum in pneumonia, pulmonary edema, 
and tuberculosis. It is usually absent in bronchitis. 
A test for albumin may, therefore, be of great value in 
distinguishing between bronchitis and tuberculosis, a 
negative result practically proving the absence of tuber- 
culosis. It is carried out as follows: The sputum is 
acidified with acetic acid to precipitate mucin and fil- 



64 THE SPUTUM 

tered. If tenacious, it is first shaken up with water. 
The filtrate is then tested for albumin, as described in the 
chapter upon the Urine. 

IV. THE SPUTUM IN DISEASE 

Strictly speaking, any appreciable amount of sputum 
is abnormal. A great many healthy persons, however, 
raise a small quantity each morning, owing chiefly to 
the irritation of inhaled dust and smoke. Although 
not normal, this can hardly be spoken of as pathologic. 
It is particularly frequent in city dwellers and in those 
who smoke cigarettes to excess. In the latter, the 
amount is sometimes so great as to arouse suspicion of 
tuberculosis. Such '' normal morning sputum " gen- 
erally consists of small, rather dense, mucoid masses, 
translucent white, or, when due to inhaled smoke, gray 
in color. Microscopically, there are a few pus-cor- 
puscles, and, usually, many alveolar cells, both of which 
may contain carbon particles. The alveolar cells com- 
monly show myelin degeneration, and free myeHn glob- 
ules may be present in large numbers. Saprophytic 
bacteria may be present, but are not abundant. 

1. Acute Bronchitis. — There is at first a small amount 
of tenacious, almost purely mucoid sputum, frequently 
blood streaked. This gradually becomes more abun- 
dant, mucopurulent in character, and yellowish or gray 
in color. At first the microscope shows a few leuko- 
cytes and alveolar and bronchial cells; later, the leuko- 
cytes become more numerous. Bacteria are not usually 
abundant. 

2. Chronic Bronchitis.— The sputum is usually abun- 
dant, mucopurulent, and yellowish or yellowish-green in 



THE SPUTUM IN DISEASE 65 

color. Nummular masses — circular, " coin-like " discs 
which sink in water — may be seen. Microscopically, 
there are great numbers of leukocytes, often much degen- 
erated. Epithelium is not abundant. Bacteria of 
various kinds, especially staphylococci, are usually nu- 
merous. 

In fibrinous bronchitis there are found, in addition, 
fibrinous casts, usually of medium size. 

In the chronic bronchitis accompanying long-continued 
passive congestion of the lungs, as in poorly compensated 
heart disease, the sputum may assume a rusty brown 
color, owing to presence of large numbers of the " heart- 
failure cells " previously mentioned. 

3. Bronchiectasis.— When there is a single large 
cavity, the sputum is very abundant at intervals, — 
sometimes as high as a liter in twenty-four hours, — and 
has a very offensive odor. It is thinner than that of 
chronic bronchitis, and upon standing separates into 
three layers of pus, mucus, and frothy serum. It 
contains great numbers of miscellaneous bacteria. 

4. Gangrene of the Lung.— The sputum is abun- 
dant, fluid, very offensive, and brownish in color. It 
separates into three layers upon standing^a brown 
deposit, a clear fluid, and a frothy layer. Microscopic- 
ally, few cells of any kind are found. Bacteria are ex- 
tremely numerous; among them may sometimes be 
found an acid-fast bacillus probably identical with the 
smegma bacillus. As before stated, elastic fibers are 
usually present in large fragments. 

5. Pulmonary Edema.— Here there is an abundant, 
watery, frothy spfutum, varying from faintly yellow or 
pink to dark brown in color; a few leukocytes and 

5 



66 THE SPUTUM 

epithelial cells and varying numbers of red blood-cor- 
puscles are found with the microscope. 

6. Bronchial Asthma.— The sputum during and fol- 
lowing an attack is scanty and very tenacious. Most 
characteristic is the presence of Curschmann's spirals, 
Charcot-Leyden crystals, and eosinophilic leukocytes. 

7. Croupous Pneumonia.— Characteristic of this dis- 
ease is a scanty, rusty red, very tenacious sputum, con- 
taining red corpuscles or altered blood-pigment, leuko- 
cytes, epithelial cells, usually many pneumococci, and 
often ver}' small fibrinous casts. This sputum is seen 
during the stage of red hepatization. During resolu- 
tion the sputum assumes the appearance of that of chronic 
bronchitis. When pneumonia occurs during the course 
of a chronic bronchitis, the characteristic rusty red 
sputum may not appear. 

8. Pulmonary Juberculosis.^The sputum is varia- 
ble. In the earliest stages it may be scanty and almost 
purely mucoid, with an occasional yellow flake, or there 
may be only a very small mucopurulent mass. When 
the quantity is very small, there may be no cough, the 
sputum reaching the larynx by action of the bronchial 
cilia. This is not well enough recognized by practi- 
tioners. A careful inspection of all the sputum brought 
up by the patient on several successive days, and a 
microscopic examination of all yellow portions, will not 

"infrequently establish a diagnosis of tuberculosis when 
physical signs are negative. Tubercle bacilli will some- 
times be found in large numbers at this stage. Blood- 
streaked sputum is strongly suggestive of tuberculosis, 
and is more common in the early stages than later. 
The sputum of more advanced cases resembles that of 



THE SPUTUM IN DISEASE 67 

chronic bronchitis, with the addition of tubercle bacilli 
and elastic fibers. Caseous particles containing im- 
mense numbers of the bacilli are common. Far- 
advanced cases with large cavities often show rather 
firm, spheric or ovoid masses of thick pus in a thin fluid 
— the so-called " globular sputum." These globular 
masses usually contain many tubercle bacilli. Con- 
siderable hemorrhages are not infrequent, and for some 
time thereafter the sputum may contain clots of blood 
or be colored brown. 



CHAPTER II 

THE URINE 

Preliminary Considerations. — The urine is an aqueous 
solution of various organic and inorganic substances. 
It is probably both a secretion and an excretion. Most 
of the substances in solution are either waste-products 
from the body metabolism or products derived directly 
from the foods eaten. Normally, the total amount of 
solid constituents carried off in twenty-four hours is about 
60 gm., of which the organic substances make up about 
35 gm. and the inorganic about 25 gm. 

The most important organic constituents are urea, 
uric acid, and ammonia. Urea constitutes about one- 
half of all the solids, or about 30 gm. in twenty-four 
hours. 

The chief inorganic constituents are the chlorids, 
phosphates, and sulphates. The chlorids, practically 
all in the form of sodium chlorid , make up about one-half 
of the inorganic substances, or about 13 gm., in twenty- 
four hours. 

Certain substances appear in the urine only in patho- 
logic conditions. The most important of these are pro- 
teins, sugars, acetone, and related substances, bile, hemo- 
globin, and the diazo substances. 

In addition to the substances in solution all urines 
contain various microscopic structures. 

While, under ordinary conditions, the composition of 

68 



THE URINE 69 

urine does not vary much from day to day, it varies 
greatly at different hours of the same day. It is evident, 
therefore, that no quantitative test can be of value unless a 
sample of the mixed twenty-four-hour urine be used. The 
patient should be instructed to void all the urine during 
the twenty-four hours into a clean vessel kept in a cool 
place, to mix it well, to measure the whole quantity, and 
to bring eight or more ounces for examination, A pint 
fruit-jar is a convenient container. When it is desired 
to make only qualitative tests, as for albumin or sugar, a 
" sample " voided at random will answer. It should be 
remembered, however, that urine passed about three 
hours after a meal is most likely to contain pathologic 
substances. That voided first in the morning is least 
likely to contain them. To diagnose cyclic albuminuria 
samples obtained at various periods during the twenty- 
four hours must be examined. 

The urine must be examined while fresh. Decom- 
position sets in rapidly, especially in warm weather, and 
greatly interferes with all the examinations. Decom- 
position may be delayed by adding five grains of boric 
acid (as much of the powder as can be heaped upon a 
ten-cent piece) for each four ounces of urine. Formalin, 
in proportion of one drop to four ounces, is also an effi- 
cient preservative, but if larger amounts be used, it may 
give reactions for sugar and albumin, and is likely to 
cause a precipitate which greatly interferes with the 
microscopic examination. 

Normal and abnormal pigments, which interfere with 
certain of the tests, can be removed by filtering the urine 
through animal charcoal, or precipitating with a solu- 
tion of acetate of lead and filtering. 



70 THE URINE 

Certain cloudy urines cannot be clarified by ordinary 
filtration through paper, particularly when the cloudi- 
ness is due to bacteria. Such urines can usually be 
rendered perfectly clear by adding a small amount of 
purified talc, shaking well, and filtering. 

A suspected fluid can be identified as urine by detect- 
ing any considerable quantity of urea in it (p. 93). 
Traces of urea may, however, be met with in ovarian cyst 
fluid, while urine from very old cases of hydronephrosis 
may contain little or none. 

The frequency of micturition is often suggestive in 
diagnosis. Whether it is unduly frequent can best be 
ascertained by asking the patient whether he has to get 
up at night to urinate. Increased frequency may be 
due to restlessness; to increased quantity of urine; to 
irritability of the bladder, usually an evidence of cys- 
titis; to obstruction (" retention with overflow"); or to 
paralysis of the sphincter. 

Clinical examination of the urine may conveniently 
be considered under four heads: I. Physical examina- 
tion. 11. Chemic examination. III. Microscopic ex- 
amination. IV. The urine in disease. 

I. PHYSICAL EXAMINATION 

1. Quantity. — The quantity passed in twenty-four 
hours varies greatly with the amount of liquids ingested, 
perspiration, etc. The normal may be taken as 1000 to 
1500 c.c, or 40 to 50 ounces. 

The quantity is increased (polyuria) during absorption 
of large serous efTusions and in many nervous conditions. 
It is usually much increased in chronic interstitial nephri- 
tis, diabetes insipidus, and diabetes melHtus. In these 



PHYSICAL EXAMINATION 7 1 

conditions a permanent increase in amount of urine is 
characteristic — a fact of much value in diagnosis. In 
diabetes mellitus the urine may, though rarely, reach the 
enormous amount of 50 liters. 

The quantity is decreased (oliguria) in severe diarrhea ; 
in fevers; in all conditions which interfere with circula- 
tion in the kidney, as poorly compensated heart disease ; 
and in the parenchymatous forms of nephritis. In 
uremia the urine is usually very greatly decreased and 
may be entirely suppressed (anuria). 

2. Color. — This varies considerably in health, and 
depends largely upon the quantity of urine voided. The 
usual color is yellow or reddish-yellow, due to the pres- 
ence of several pigments, chiefly urochrome. Acid urine 
is generally darker than alkaline. In recording the 
color Vogel's scale (see Frontispiece) is very widely used, 
the urine being filtered and examined by transmitted 
light in a glass three or four inches in diameter. 

The color is sometimes greatly changed by abnormal 
pigments. Blood-pigment gives a red or brown, smoky 
color. Urine containing bile is yellowish or brown, with 
a yellow foam when shaken. It may assume a greenish 
hue after standing, owing to oxidation of bilirubin into 
biliverdin. Ingestion of small amounts of methylene- 
blue gives a pale green; large amounts give a marked 
blue. Santonin produces a yellow; rhubarb, senna, 
cascara, and some other cathartics, a brown color; 
these change to red upon addition of an alkali, and if 
the urine be alkaline when voided, may cause suspicion 
of hematuria. Thymol gives a yellowish-green. Fol- 
lowing poisoning from phenol and related drugs the urine 
may have a normal color when voided, but becomes olive- 



72 THE URINE 

green to brownish-black upon standing. In susceptible 
individuals therapeutic doses of creosote, or absorption 
from carbolized dressings, may cause this change. Urine 
which contains melanin, as sometimes in melanotic 
sarcoma, and very rarely in wasting diseases, also be- 
comes brown or black upon long standing. A similar 
darkening upon exposure to the air occurs in alkapto- 
nuria (p. 126). 

A pale greenish urine with high specific gravity 
strongly suggests diabetes mellitus. 

3. Transparency. — Freshly passed normal urine is 
clear. Upon standing, a faint cloud of mucus, leuko- 
cytes, and epithelial cells settles to the bottom — the so- 
called "nubecula." Abnormal cloudiness is usually due 
to presence of phosphates, urates, pus, blood, or bacteria. 

Amorphous pJiosphates are precipitated in neutral or 
alkaline urine. They form a white cloud and sediment, 
which disappear upon addition of an acid. 

Amorphous urates are precipitated only in acid urine. 
They form a white or pink cloud and sediment (" brick- 
dust deposit "), which disappear upon heating. 

Pus resembles amorphous phosphates to the naked eye. 
Its nature is easily recognized wdth the microscope, oi" 
by adding a strong solution of caustic soda to the sedi- 
ment, which is thereby transformed into a gelatinous 
mass (Donne's test). 

Blood gives a reddish or brown, smoky color, and may 
be recognized with the microscope or by tests for hemo- 
globin. 

Bacteria, when present in great numbers, give a uni- 
form cloud, which cannot be removed by ordinary filtra- 
tion. They are detected with the microscope. 



PHYSICAL EXAMINATION 73 

The cloudiness of decomposing urine is due mainly to 
precipitation of phosphates and multiplication of bac- 
teria. 

4. Odor. — The characteristic aromatic odor is due to 
volatile acids, and is most marked in concentrated urines. 
During decomposition, the odor becomes ammoniacal. 
A fruity odor is sometimes noted in diabetes, due prob- 
ably to acetone. Urine which contains cystin may de- 
velop an odor of sulphureted hydrogen during decom- 
position. 

Various articles of diet and drugs impart peculiar 
odors. Notable among these are asparagus, which gives 
a characteristic oflfensive odor, and turpentine, which 
imparts an odor somewhat suggesting that of violets. 

5. Reaction. — Normally, the mixed twenty-four-hour 
urine is slightly acid in reaction. The acidity was 
formerly held to be due to acid phosphates, but Folin 
has shown that the acidity of a clear urine is ordinarily 
much greater than the acidity of all the phosphates, the 
excess being due to free organic acids. Individual 
samples may be slightly alkaline, especially after a full 
meal, or amphoteric. The reaction is determined by 
means of htmus-paper. 

Acidity is increased after administration of certain 
drugs, and whenever the urine is concentrated from any 
cause, as in fevers. A very acid urine may cause fre- 
quent micturition because of its irritation. This is often 
an important factor in the troublesome enuresis of 
children. 

The urine always becomes alkahne upon long standing, 
owing to decomposition of urea with formation of am- 
monia. If markedly alkaline when voided, it usually 



74 THE URINE 

indicates such " ammoniacal decomposition " in the 
bladder, which is the rule in chronic cystitis, especially 
that due to paralysis or obstruction. Alkalinity due to 
ammonia (volatile alkalinity) can be distinguished by 
the fact that litmus paper turned blue by the urine again 
becomes red upon gentle heating. Fixed alkalinity is 
due to alkaline salts, and is often observ^ed during fre- 
quent vomiting, after the crisis of pneumonia, in various 
forms of anemia, after full meals, and after administra- 
tion of certain drugs, especially salts of vegetable acids. 
Quantitative estimation of the acidity of urine is not 
of much clinical value. When, however, it is desired 
to make it, the method of Folin will be found satis- 
factory. In every case the sample must be from the 
mixed twenty-four-hour u^ine and as fresh as possible. 

Folin's Method. — Into a small flask measure 25 c.c. of 
the urine and add i or 2 drops 0.5 per cent, alcoholic solu- 
tion of phenolphthalein and 15 or 20 gm. of neutral potassium 
oxalate. Shake for a minute, and immediately titrate with 
decinormal sodium hydroxid, shaking meanwhile, until the 
first permanent pink appears. Read off from the buret the 
amount of decinormal sodium hydroxid solution added, and 
calculate the number of cubic centimeters which would be 
required for the entire twenty-four hours' urine. Folin 
places the normal acidity, obtained in this way, at 617. 

6. Specific Gravity— The normal average is about 
1. 01 7 to 1.020. Samples of urine taken at random may 
go far above or below these figures, hence a sample of the 
mixed twenty-four-hour urine should always be used. 

Pathologically, it may vary from i.ooi to 1.060. It is 
low in chronic interstitial nephritis, diabetes insipidus, 



PHYSICAL EXAMINATION 



75 



and many functional nervous disorders. It is high in 
fevers and in parenchymatous forms of nephritis. In 
any form of nephritis a sudden fall without a corre- 
sponding increase in quantity of urine may foretell ap- 
proaching uremia. It is highest in diabetes melUtus. A 
high specific gravity when the urine is not highly colored 
should lead one to suspect this disease. A normal 
specific gravity does not, however, exclude it. 




l! 



I 



Fig. 17. — Squibb's urinometer with thermometer and cylinder. 



The specific gravity is most conveniently estimated by 
means of the urinometer — Squibb's is preferable (Fig. 
17). It is standardized for a temperature of 77° F., and 
the urine should be at or near that temperature. Care 
should be taken that the urinometer does not touch the 
side of the tube, and that air-bubbles are removed from 
the surface of the urine. With most instruments the 
reading is taken from the bottom of the meniscus. A 
long scale on the stem is desirable, because of the greater 
ease of accurate reading. 



76 



THE URINE 



One frequently wishes to ascertain the specific gravity 
of quantities of fluid too small to float an urinometer. 
A simple device for this purpose, which requires only 
about 3 c.c. and is very satisfactory in clinical work has 
been designed by Saxe (Fig. i8). The urine is placed in 
the bulb at the bottom, the instrument is floated in dis- 




Fig. i8. — Saxe's urinopyknometer and jar for same. 

tilled water, and the specific gravity is read off from the 
scale upon the stem. 

7. Total Solids. — An estimation of the total amount 
of solids which pass through the kidneys in twenty-four 
hours is, in practice, one of the most useful of urinary 
examinations. The normal for a man of 150 pounds is 
about 60 gm., or 950 gr. The principal factors which 



PHYSICAL EXAMINATION 77 

influence this amount are body weight (except with 
excessive fat), diet, exercise, and age, and these should 
be considered in making an estimation. After about 
the f orty-fif th year it becomes gradually less ; after 
seventy-five years it is about one-half the amount given. 

In disease, the amount of solids depends mainly upon 
the activity of metabolism and the ability of the kidneys 
to excrete. An estimation of the solids, therefore, 
furnishes an important clue to the functional efficiency 
of the kidneys. The kidneys bear much the same 
relation to the organism as does the heart: they cause 
no direct harm so long as they are capable of perform- 
ing the work required of them. When, however, through 
either organic disease or functional inactivity, they fail 
to carry off their proportion of the waste-products of the 
body, some of these products must either be eliminated 
through other organs, where they cause irritation and 
disease, or be retained within the body, where they act 
as poisons. The great importance of these poisons in 
production of distressing symptoms and even organic 
disease is not well enough recognized by most practi- 
tioners. Disappearance of unpleasant and perplexing 
symptoms as the urinary solids rise to the normal under 
proper treatment is often most surprising. 

When, other factors remaining unchanged, the 
amount of solids eliminated is considerably above the 
normal, increased destructive metabolism may be 
inferred. 

The total solids can be estimated roughly, but ac- 
curately enough for most clinical purposes, by multi- 
plying the last two figures of the specific gravity of the 
mixed twenty-four-hour urine by the number of ounces 



78 THE URINE 

voided and to the product adding one-tenth of itself. 
This gives the amount in grains. If, for example, the 
twenty-four-hour quantity is 3 pints or 48 ounces, and 
the specific gravity is 1.018, the total solids would ap- 
proximate 950 gr., as follows: 

48 X 18 = 864; 864 + 86.4 - 950.4 

This method is especially convenient for the practi- 
tioner, because patients nearly always report the amount 
of urine in pints and ounces, and it avoids the necessity 
of converting into the metric system. Haser's method is 
more widely used but is less convenient. The last two 
figures of the specific gravity are multiplied by 2.33. 
The product is then multiplied by the number of cubic 
centimeters voided in twenty-four hours and divided 
by 1000. This gives the total solids in grams. 

8. Functional Tests.— Within the past few years 
much thought has been devoted to methods of more 
accurately ascertaining the functional efficiency of the 
kidneys, especially of one kidney when removal of the 
other is under consideration. The most promising of 
the methods which have been devised are cryoscopy, 
electric conductivity, the methylene-blue test, and the 
phloridzin test. It is doubtful whether, except in 
experienced hands, these yield any more information 
than can be had from an intelligent consideration of the 
specific gravity and the twenty-four-hour quantity, to- 
gether wath a microscopic examination. They are most 
useful when the urines obtained from separate kidneys 
by segregation or ureteral catheterization are compared. 
The reader is referred to larger works upon urinalysis 
for details. 



PHYSICAL EXAMINATION 79 

Cryoscopy, determination of the freezing-point, de- 
pends upon the principle that the freezing-point of a 
fluid is depressed in proportion to the number of mole- 
cules, organic and inorganic, in solution. To have any 
value, the freezing-point of the urine must be compared 
with that of the blood, since it is not so much the num- 
ber of molecules contained in the urine as the number 
which the kidney has failed to carry off and has left in 
the blood, that indicates its insufficiency. 

Electric conductivity refers to the power of the urine 
to carry an electric current. It is increased in pro- 
portion to the nimiber of inorganic molecules in solution. 

In the methylene-blue test of Achard and Castaigne 
a solution of methylene-blue is injected intramuscularly, 
and the time of its appearance in the urine is noted. 
Normally, it appears in about thirty minutes. When 
delayed, renal " permeability " is supposed to be inter- 
fered with. Since methylene-blue is sometimes ex- 
creted as a colorless derivative, indigo-carmin has been 
proposed as a substitute. In the absence of renal in- 
sufficiency this always gives a blue color, which begins 
to appear in about five minutes. 

The phloridzin test consists in the hypodermic injec- 
tion of a small quantity of phloridzin. This substance 
is transformed into glucose by the kidneys of healthy 
persons. In disease, this change is more or less inter- 
fered with, and the amount of glucose recoverable from 
the urine is taken as an index of the secretory power of 
the kidneys. 

In applying these tests for " permeability," " secre- 
tory ability," etc., one must rertiember that the condi- 
tions are abnormal, and that there is no e\ddence that 



8o THE LTIIXE 

the kidneys will behave with the products of metabolism 
as they do with the substances selected for the tests, and 
also that the tests throw unusual work upon the kidneys, 
which in some cases may be harmful. 

II. CHEMIC EXAMINATION 

A. Normal Constituents 

Of the large number of organic and inorganic sub- 
stances normally present in the urine, only a few demand 
any consideration from the clinician. The following 
table, therefore, outlines the average composition from 
the clinical, rather than from the chemical, standpoint. 
Only the twenty-four-hour quantities are given, since 
they alone furnish an accurate basis for comparison. 
The student tannot too soon learn that percentages mean 
little or nothing, excepting as they furnish a means of 
calculating the twenty-four-hour elimination. 

COMPOSITION OF NORMAL URINE 

Grams in twenty- Approximate 

four hours. average. 

Total substances in solution 55-70 60 

Inorganic substances 20-30 25 

Chlorids (chiefly sodium chlorid) 10-15 12.5 

Phosphates (estimated as phosphoric acid), 

total 2.5-3.5 3 

Earthy 3 of total i 

Alkaline § of total 2 

Sulphates (estimated as sulphuric acid), total 1.5-3.0 2.5 

Mineral j% of total 2.25 

Conjugate yV of total 0.25 

Includes indiran Trace 

Ammonia 0.5-1 .0 0.7 

Organic substances 30-40 35 

Urea 20-35 3° 

Uric acid 0.4-1.0 0.7 



CHEMIC EXAMINATION 



8l 




Fig. iQ. — Graphic expression of quantities in the urine. Solid line, normal urine; dotted 
line, an example of pathologic urine in a case of cancerous cachexia (Saxe). 



82 THE URINE 

Although the conjugate sulphates are organic com- 
pounds, they are, for the sake of convenience, included 
with the inorganic sulphates in the table on p. 80. 

Among constituents which are of little clinical im- 
portance, or are present only in traces, are: 

Inorganic: Iron, carbonates, nitrates, silicates, and 
fiuorids. 

Organic: Creatinin, hippuric acid, purin bases, oxalic 
acid, benzoic acid, volatile fatty acids, pigments, and 
acetone. 

Variations in body weight, diet, and exercise cause 
marked fluctuations in the total solids and in individual 
substances. 

1. Chlorids. — These are derived from the food, and 
are mainly in the form of sodium chlorid. The amount 
excreted normally is 10 to 15 gm. in twenty-four hours. 
It is much affected by the diet, and is reduced to a 
minimum in starvation. 

Excretion of chlorids is diminished in nephritis and 
in fevers, especially in pneumonia and inflammations 
leading to the formation of large exudates. In nephritis 
the kidneys are less permeable to the chlorids, and it is 
possible that the edema is due largely to an effort of 
the body to dilute the chlorids which have been retained. 
Certainly an excess of chlorids in the food will increase 
both the albuminuria and the edema of nephritis. In 
fevers the diminution is due largely to decrease of food, 
though probably in some measure to impaired renal 
function. In pneumonia chlorids are constantly very 
low, and in some cases are absent entirely. Following 
the crisis they are increased. In inflammations leading 
to formation of large exudates — e. g., pleurisy with 



CHEMIC EXAMINATION 



83 



effusion — chlorids are diminished, because a consider- 
able amount becomes " locked up " in the exudate. 
During absorption chlorids are liberated and appear 
in the urine in excessive amounts. 

Diminution of chlorids is also observed in severe 
diarrhea, anemic conditions, and carcinoma of the 
stomach. 




Fig. 20.— The Purdy electric centrifuge with four arras. 

Quantitative Estimation. — The best method for clinical 
purposes is the centrifugal method. 

Purdy's Centrifugal Methods. — As shown by the late 
Dr. Purdy, the centrifuge offers an important means 
of making quantitative estimations of a number of sub- 
stances in the urine. Results are easily and quickly 
obtained, and are probably accurate enough for all clini- 
cal purposes. 

In general, the methods consist in precipitating the 
substance to be estimated in a graduated centrifuge tube. 



84 



THE URINE 



and applying a definite amount of centrifugal force for a 
definite length of time, after which the percentage of 
precipitate is read off upon the side of the tube. Al- 
bumin, if present, must be previously removed by boil- 
ing and filtering. Results are in terms of bulk of pre- 
cipitate, which must not be confused with percentage 
by weight. The weight percentage can be found by 
referring to Purdy's tables, given later. In this, as in 




Fis- 21. — Water-motor centrifuge. 



all quantitative urine work, percentages mean little in 
themselves; the actual amount eliminated in twenty- 
four hours should always be calculated. 

The centrifuge should have an arm with radius of 6f 
inches when in motion, and should be capable of main- 
taining a speed of 1500 revolutions a minute. The 
electric centrifuge is to be recommended, although 
good work can be done with a water-power centrifuge, 
or, after a little practice, with the hand centrifuge. A 



CHEMIC EXAMINATION 



85 



speed indicator is desirable with electric and water- 
motor machines, although one can learn to estimate the 
speed by the musical note. 



c.c\ 




Fig. 22. — Purdy's tubes for the centrifuge: a, Percentage tube; 6, sediment tube. 



Estimation of Chlorids. — Fill the graduated tube to the 
10 CO. mark with urine; add 15 drops strong nitric acid and 
then silver nitrate solution (dram to the ounce) to the 15 c.c, 
mark. Mix by inverting several times. Let stand a few 
minutes for a precipitate to form, and then revolve in the 
centrifuge for three minutes at 1200 revolutions a minute. 
Each one-tenth cubic centimeter of precipitate equals i per 
cent, by bulk. The normal is about 10 per cent. This may 
be converted into terms of chlorin or sodium chlorid by means 
of the table upon page 86. Roughly speaking, the percent- 



86 



THE URINE 



age of chlorin by weight is about one-twelfth the bulk- 
percentage. 

TABLE FOR THE ESTIMATION OF CHLORIDS AFTER 
CENTRIFUGATION 

Showing the bulk-percentage of silver chlorid {AgCl) and the correspond- 
ing gravimetric percentages and grains per fluidounce of sodium chlorid 
{NaCl) and chlorin {CI). — (Ptirdy.) 





d 

C3 


u 

a 


d 


u 







y 


u 





2 


C 


"A 

N 

1-1 


V 

s 

a 




SO 

.', 


V 

be 

s 


K 




g 




Ji 


^ 
b 







iZ 




3 


1 


c 




1^ 




i 


0.03 


0.15 


0.02 


0.1 1 


8 


1.04 


4.98 


0.63 


3.02 


i 


0.07 


0.31 


0.04 


0.19 


82 


I.I 


5-29 


0.67 


3.22 


1 


O.I 


0.47 


0.06 


0.28 


9, 


1. 17 


5-6 


0.71 


3-4 


I 


0.13 


0.62 


0.08 


0.38 


9^ 


1.23 


5-91 


0-75 


3-6 


i\ 


0.16 


0.78 


O.I 


0.48 


10 


1-3 


6.22 


0.79 


3-79 


li 


0.19 


0-93 


0.12 


0-57 


10^ 


1.36 


6.53 


0.83 


3-97 


i| 


0.23 


1.09 


0.14 


0.67 


II 


1-43 


6.84 


0.87 


4.16 


2 


0.26 


1.24 


0.16 


0.76 


iij 


1.49 


7.2 


0.91 


4-35 


2^ 


0.29 


1.41 


0.18 


0.85 


12 


1.56 


7.46 


0-95 


4-54 


2j 


0.32 


1.56 


0.2 


0.96 


12J 


1.62 


7.78 


0.99 


4-73 


2| 


0.36 


1.71 


0.22 


1.04 


13, 


1.69 


8.09 


1.02 


4.92 


3 


0-39 


1.87 


0.24 


1-13 


13J 


1-75 


8.4 


1.06 


5-II 


3} 


0.42 


2.02 


0.26 


1.23 


14 


1.82 


8.71 


I.I 


529 


4 


0.4S 


2.18 


0.28 


1.32 


14J 


1.88 


9.02 


1. 14 


5-49 


3l 


0.49 


2-35 


0-3 


1.42 


15^ 


1.94 


9-33 


1. 18 


5-67 


4 


0.52 


2.49 


0.32 


1-51 


15^ 


2.01 


965 


1.22 


5-86 


4} 


0-55 


2.64 


0-34 


1.61 


16 


2.07 


9.94 


1.26 


6.06 


4* 


0.58 


2.8 


0-3S 


1-7 


16J 


2.14 


10.27 


1-3 


6.24 


4l 


0.62 


2.96 


0-37 


1.8 


'7, 


2.2 


10.151 


1-34 


6.43 


5, 


0.65 


3-II 


0-39 


1.89 


17J 


2.27 


10.87 


1.38 


6.62 


5h 


0.71 


3-42 


0.43 


2.09 


18 


2-33 


II. 2 


1.42 


6.81 


6 


0.78 


3-73 


0.47 


2.27 


18J 


2.4 


11.51 


1.46 


7.0 


6J 


0.84 


4-05 


0.51 


2.46 


19 


2.46 


11.82 


i-S 


7.19 


7, 


0.91 


4-35 


0-S5 


2.62 


19^ 


2-53 


12.13 


1-54 


7-38 


7i 


0.97 


4.67 


0-59 


2.84 


20 


2-59 


12.44 


1.58 


7-56 



Bulk-percentage to be read on the side of the tube. 



2. Phosphates. — Phosphates are derived largely from 
the food, only a small proportion resulting from metab- 



CHEMIC EXAMINATION 87 

olism. The normal daily output of phosphoric acid is 
about 2.5 to 3.5 gm. 

The urinary phosphates are of two kinds: alkaline, 
which make up two-thirds of the whole, and include the 
phosphates of sodium and potassium; and earthy, which 
constitute one-third, and include the phosphates of cal- 
cium and magnesium. Earthy phosphates are fre- 
quently thrown out of solution in neutral and alkaline 
urines, and as " amorphous phosphates " form a very 
common sediment. This sediment seldom indicates an 
excessive excretion of phosphoric acid. It is usually 
merely an evidence of diminished acidity of the urine, or 
of an increase in the proportion of phosphoric acid elimi- 
nated as earthy phosphates. This form of " phospha- 
turia " is most frequent in neurasthenia and hysteria. 
When the urine undergoes ammoniacal decomposition, 
some of the ammonia set free combines with magnesium 
phosphate to form ammoniomagnesium phosphate 
(" triple phosphate "), which is deposited in typical 
crystalline form (p. 148). 

Excretion of phosphates is increased by a rich diet; 
in active metabolism ; in certain nervous and mental dis- 
orders; in leukemia; and in phosphatic diabetes, an ob- 
scure disturbance of metabolism (not related to diabetes 
mellitus) which is associated with an increase in the out- 
put of phosphates up to 10 gm. or more in twenty-four 
hours. Phosphates are decreased in chronic diseases 
with lowered metabolism; in hepatic cirrhosis and acute 
yellow atrophy; in pregnancy, owing to developing 
fetal bones; and in nephritis, owing to kidney imper- 
meability. 

Quantitative estimation does not furnish much of 



88 



THE URINE 



definite clinical value. The centrifugal method is the 
most convenient. 

T.\BLE FOR THE ESTIMATION OF PHOSPHATES AFTER 
CEXTRIFUGATION 

Showing hulk-percentages of uranyi phosphate {H\UO^PO^ and the cor- 
responding gravimetric percentages and grains per ounce oj phosphoric 
acid {P.p^).—{Purdy.) 



Bulk-per- 

rentage of 

H(U0„)P04. 


Percentage 
P.O5. 


Or. Per Oz. 
P2O5. 


Bulk-per- 
centage of 
H(U0.^)P04. 

1 1 


Percentage 


Gr. Per Oz. 
P-iO,. 


b 


0.02 


O.I 


0.14 


0.67 


I 


0.04 


0.19 


12 


o.is 


0.72 


ij 


0.045 


0.22 


13 


0.16 


0.77 


2 


0.05 


0.24 


14 


0.17 


0.82 


2 1 


0-055 


0.26 


IS 


0.18 


0.86 


3, 


0.06 


0.29 


16 


0.19 


0.91 


3i 


0.065 


0.31 


17 


0.2 


0.96 


4 


0.07 


0.34 


18 


0.21 


I. 


4i 


0.075 


0.36 


19 


0.22 


1.06 


5 


0.08 


0.38 


20 


0.23 


I.I 


6 


0.09 


0.43 


21 


0.24 


115 


/ 


O.I 


0.48 


22 


0.25 


1.2 


8 


O.II 


0-53 


^3 


0.26 


1-25 


9 


0.12 


0-58 


24 


0.27 


1-3 


lO 


0.13 


0.62 1 


25 


0.28 


1-35 



Bulk-percentage to be read from graduation on the side of the tube. 

Purdy's Centrifugal Method. — Take 10 c.c. urine in the 
graduated tube, add 2 c.c. of 50 per cent, acetic acid, and 3 c.c. 
of 5 per cent, uranium nitrate solution. Mix; let stand a few 
minutes, and revolve for three minutes at 1200 revolutions. 
The bulk of precipitate is normally about 8 per cent. The 
percentage of phosphoric acid by weight is, roughly, one- 
eighty-fifth of the bulk-percentage. 

3. Sulphates. — The urinary sulphates are derived 
partly from the food, especially meats, and partly from 
body metabolism. The normal output of sulphuric acid 
is about 1.5 to 3 gm. daily. It is increased in condi- 



CHEMIC EXAMINATION 



89 



tions associated with active metabolism, and in general 
may be taken as a rough index of protein metabolism. 

Quantitative estimation of the total sulphates yields 
little of clinical value. 



Purdy's Centrifugal Method. — Take 10 c.c. urine in the 
graduated tube and add barium chlorid solution to the 
15 c.c. mark. This consists of barium chlorid, 4 parts; 
strong hydrochloric acid, i part; and distilled water, 16 
parts. Mix; let stand a few minutes, and revolve for three 
minutes at 1200 revolutions a minute. The normal bulk 
of precipitate is about 0.8 per cent. The percentage by 
weight of sulphuric acid is about one-fourth of the bulk- 
percentage. 

TABLE FOR THE ESTIMATION OF SULPHATES AFTER 
CENTRIFUGATION 
Showing the hulk-percentages of barium sulphate (^BaSO^ and the cor- 
responding gravimetric percentages and grains per fiuidounce of sul- 
phuric acid (SO3). — (Purdy.) 



Bulk-per- 
centage of 
BaSO*. 


Percentage 
SO3. 


Gr. Per Oz. 
SO3. 


Bulk-per- 
centage of 
BaS04. 


Percentage 
SO3. 


Gr. Per Oz. 
SO3. 


■ 


0.04 


0.19 


2i 


0-55 


2.64 


: ■ 


0.07 


0.34 


2i 


0.61 


2-93 


■ 


O.I 


0.48 


2| 


0.67 


3.22 


: ■ 


0.13 


0.62 


3, 


0-73 


3-5 




0.16 


0.77 


3* 


0.79 


3-79 




0.19 


0.91 


4 


0.85 


4.08 




0.22 


1.06 


3l 


0.91 


4-37 


I 


0.25 


I.I 


4 


0.97 


4.66 


li 


0.31 


1.49 


4i 


1.03 


4.94 


^ 


0-37 


1.78 


4i 


1.09 


5-23 


l| 


0-43 


2.06 


4l 


i-iS 


5-52 


2 


0.49 


2-35 


5 


1. 21 


5-8i 



Bulk-percentage to be read from graduation on the side of the tube. 

About nine-tenths of the sulphuric acid is in com- 
bination with various mineral substances, chiefly sodium, 



90 THE URINE 

potassium, calcium, and magnesium (mineral or pre- 
formed sulphates). One-tenth is in combination with 
certain aromatic substances, which are mostly products 
of albuminous putrefaction in the intestine, but are de- 
rived in part from destructive metabolism (conjugate 
or ethereal sulphates) . Among these aromatic substances 
are indol, phenol, and skatol. By far the most impor- 
tant of the conjugate sulphates and representative of the 
group is potassium indoxyl sulphate. 

Potassium indoxyl sulphate, or indican, is derived 
from indol. Indol is absorbed and oxidized into in- 
doxyl, which combines with potassium and sulphuric 
acid and is thus excreted. Under normal conditions 
the amount in the urine is small. It is increased by a 
meat diet. 

Unlike the other ethereal sulphates, which are de- 
rived in part from metabolism, indican originates prac- 
tically wholly from putrefactive processes. It alone, 
therefore, and not the total ethereal sulphates, can be 
taken as an index of such putrefaction. A pathologic 
increase is called indicanuria. It is noted in: 

(a) Diseases of the Small Intestine. — This is by far 
the most common source. Intestinal obstruction gives 
the largest amounts of indican. It is also much in- 
creased in intestinal indigestion — so-called " bilious- 
ness "; in inflammations, especially in cholera and ty- 
phoid fever; and in paralysis of peristalsis, such as 
occurs in peritonitis. Simple constipation and diseases 
of the large intestine alone do not so frequently cause 
indicanuria. 

(b) Diseases of the stomach associated with deficient 
hydrochloric acid, as chronic gastritis and gastric cancer. 



CHEMIC EXAMINATION 9 1 

Diminished hydrochloric acid favors intestinal putre- 
faction. 

(c) Diminished Flow of Bile. — Since the bile serves 
both as a stimulant to peristalsis and an intestinal anti- 
septic, a diminished flow from any cause favors occur- 
rence of indicanuria. 

{d) Decomposition of exudates anywhere in the body, 
as in empyema, bronchiectasis, and large tuberculous 
cavities. 

Detection of indican depends upon its decomposition 
and oxidation of the indoxyl set free into indigo-blue. 
This change sometimes takes place spontaneously in 
decomposing urine, causing a dirty blue color. Crystals 
of indigo (Fig. 36) may be found both in the sediment 
and the scum. 

Obermayer's Method. — In a test-tube take equal parts of 
the urine and Obermayer's reagent and add a small quantity 
of chloroform. Mix by inverting a few times; avoid shak- 
ing violently. If indican be present in excess, the chloroform, 
which sinks to the bottom, will assume an indigo-blue color. 
It will take up the indigo more quickly if the urine be warm. 
The depth of color indicates the comparative amount of 
indican if the same proportions of urine and reagents are 
always used, but one should bear in mind the total amount 
of urine voided. The indican in normal urine may give 
a faint blue by this method. Urine of patients taking iodids 
gives a reddish-violet color, which disappears upon addition 
of a few drops of strong sodium hyposulphite solution and 
shaking. Bile-pigments, which interfere with the test, must 
be removed (p. 69), 

Obermayer^s reagent consists of strong hydrochloric acid 
(sp. gr., 1. 19), 1000 parts, and ferric chlorid, 2 parts. This 
makes a yellow, fuming liquid which keeps well. 



92 THE URINE 

4. Urea. — From the standpoint of physiolog}-^ urea is 
the most important constituent of the urine. It is the 
principal waste-product of metaboHsm, and constitutes 
about one-half of all the solids excreted — about 20 to 
35 gm. in twenty-four hours. It represents 85 to 90 
per cent, of the total nitrogen of the urine, and its quan- 
titative estimation is a simple, though not very accurate, 
method of ascertaining the state of nitrogenous excretion. 

This is true, however, only in normal individuals 
upon average mixed diet. Under pathologic conditions, 
the proportion of nitrogen distributed among the various 
nitrogen-containing substances undergoes great varia- 
tion. The only accurate index of protein metabolism 
is, therefore, the total output of nitrogen, which can be 
estimated by the Kjeldahl method. The whole subject 
of " nitrogen partition " and " nitrogen equilibrium " 
(relation of excretion to intake) is an important one, but 
is out of the province of this book, since as yet it con- 
cerns the physiologic chemist more than the clinician. 

It may be helpful to state here, however, that upon a mixed 
diet the nitrogen of the urine is distributed about as follows: 
urea nitrogen, 86.9 per cent.; ammonia nitrogen, 4.4 per 
cent.; creatinin nitrogen, 3.6 per cent.; uric acid nitrogen, 
0.75 per cent.; "undetermined nitrogen," chiefly in amino 
acids, 4.3 per cent. 

Normally, the amount is greatly influenced by ex- 
ercise and diet. It is increased by copious drinking 
of water and administration of ammonium salts of 
organic acids. 

Pathologically, urea is increased in fevers, in diabetes, 
and especially during resolution of pneumonia and ab- 



CHEMIC EXAMINATION 93 

sorption of large exudates. As above indicated, when 
other factors are equal, the amount of urea indicates 
the activity of metabolism. In deciding whether in a 
given case an increase of urea is due to increased metab- 
ohsm the relation between the amounts of urea and of 
the chlorids is a helpful consideration. The amount 
of urea is normally about twice that of the chlorids. If 
the proportion is much increased above this, increased 
tissue destruction may be inferred, since other condi- 
tions which increase urea also increase chlorids. 

Urea is decreased in diseases of the liver with destruc- 
tion of liver substance, such as cirrhosis, carcinoma, 
and acute yellow atrophy. It may or may not be 
decreased in nephritis. In the early stages of chronic 
nephritis, when diagnosis is difficult, it is usually normal. 
In the late stages, when diagnosis is comparatively easy, 
it is decreased. Hence estimation of urea is of little help 
in the diagnosis of this disease, especially when, as is so 
frequently the case, a small quantity of urine taken at 
random is used. When, however, the diagnosis is 
established, estimations made at frequent intervals 
under the same conditions of diet and exercise are of 
much value, provided a sample of the mixed twenty-four- 
hour urine he used. A steady decline is a very bad prog- 
nostic sign, and a sudden marked diminution is usually 
a forerunner of uremia. 

The presence of urea can be shown by allowing a few 
drops of the fluid partially to evaporate upon a slide, and 
adding a small drop of pure, colorless nitric acid or 
saturated solution of oxalic acid. Crystals of urea 
nitrate or oxalate (Fig. 23) will soon appear and can be 
recognized with the microscope. 



94 



THE URINE 



Quantitative Estimation. — The hypobromite method, 
which is generally used, depends upon the fact that 
urea is decomposed by sodium hypobromite with libera- 
tion of nitrogen. The amount of urea is calculated from 




Fig. 23. — Crystals of nitrate of urea (upper half) and 
oxalate of urea (lower half) (after Funke). 



Fig. 24-- 



-Doremus-Hinds' ure- 
ometer. 



the volume of nitrogen set free. The improved Doremus 
apparatus (Fig. 24) is the most convenient. 

Pour some of the urine into the smaller tube of the appa- 
ratus, then open the stopcock and quickly close it so as to fill 
its lumen with urine. Rinse out the larger tube with water 
and fill it and the bulb with 25 per cent, caustic soda solu- 
tion. Add to this i c.c. of bromin by means of a medicine- 
dropper and mix well. This prepares a fresh solution of 
sodium hypobromite with excess of caustic soda, which serves 
to absorb the carbon dioxid set free in the decomposition 
of urea. When handling bromin, keep an open vessel of 
ammonia near to neutralize the irritant fumes. 

Pour the urine into the smaller tube, and then turn the 
stopcock so as to let as much urine as desired (usually i c.c.) 



CHEMIC EXAMINATION 95 

run slowly into the hypobromite solution. When bubbles 
have ceased to rise, read off the height of the fluid in the large 
tube by the graduations upon its side. This gives the amount 
by weight of urea in the urine added, from which the amount 
excreted in twenty-four hours can easily be calculated. If 
the urine contains much more than the normal amount, it 
should be diluted. 

To avoid handling pure bromin, which is disagreeable. 
Rice's solutions may be employed: 

(a) Bromin, 31 gm. 
Potassium bromid, 31 
Distilled water, 250 c.c. 

(b) Caustic soda, 100 gm. 
Distilled water, 250 c.c. 

One part of each of these solutions and two parts of water 
are mixed and used for the test. The bromin solution must 
be kept in a tightly stoppered bottle or it will rapidly lose 
strength. 

5. Uric Acid. — Uric acid is the most important of a 
group of substances, called purin bodies, which are de- 
rived cliiefiy from the nucleins of the food and from 
metabolic destruction of the nuclei of the body. The 
daily output of uric acid is about 0.4 to i gm. The 
amount of the other purin bodies together is about 
one-tenth that of uric acid. Excretion of these sub- 
stances is greatly increased by a diet rich in nucleins, as 
sweetbreads and liver. 

Uric acid exists in the urine in the form of urates, 
which in concentrated urines are readily thrown out of 
solution and constitute the familiar sediment of " amor- 
phous urates." This, together with the fact that uric 



96 



THE URINE 






\.B9 
915 
2.45 



-fffl-s 



acid is frequently deposited as crystals, 
constitutes its chief interest to the prac- 
titioner. It is a very common error to 
consider these deposits as evidence of 
excessive excretion. 

Pathologically, the greatest increase 
of uric acid occurs in leukemia, where 
there is extensive destruction of leuko- 
cytes, and in diseases with active de- 
struction of the liver and other organs 
rich in nuclei. Uric acid is decreased 
before an attack of gout and increased 
during it, but its etiologic relation is 
still uncertain. An increase is also noted 
in acute articular rheumatism during the 
febrile stage. 

Quantitative Estimation. — The follow- 
ing are the best methods for ordinary 
clinical purposes, although no great ac- 
curacy can be claimed for them. 

Cook's Method for Purin Bodies. — In a 
centrifuge tube take lo c.c. urine and add 
about I gm. (about i c.c.) sodium car- 
bonate and I or 2 c.c. strong ammonia. 
Shake until the soda is dissolved. The 
earthy phosphates will be precipitated. 
Centrifugalize thoroughly and pour off all 
the clear fluid into a graduated centrifuge 
tube. Add 2 c.c. ammonia and 2 c.c. am- 
moniated silver nitrate solution. Let stand 
a few minutes, and revolve in the centri- 
fuge until the bulk of precipitate remains 
constant. Each one-tenth cubic centimeter 



CHEMIC EXAMINATION 97 

of sediment represents 0.001176 gm. purin bodies. This 
amount may be regarded as uric acid, since this substance 
usually constitutes nine-tenths of the purin bodies and the 
clinical significance is the same. 

Ammoniated silver nitrate solution is prepared by dissolving 
5 gm. of silver nitrate in 100 c.c. distilled water, and adding 
ammonia until the solution clouds and again becomes clear. 

Ruhemann's Method for Uric Acid. — The urine must 
be slightly acid. Fill Ruhemann's tube (Fig. 25) to the 
mark S with the indicator, carbon disulphid, and to the mark 
J with the reagent. The carbon disulphid will assume a 
violet color. Add the urine, a small quantity at a time, 
closing the tube with the glass stopper and shaking vigor- 
ously after each addition, until the disulphid loses every 
trace of its violet color and becomes pure white. This com- 
pletes the test. The figure in the right-hand column of 
figures corresponding to the top of the fluid gives the amount 
of uric acid in parts per thousand. The presence of diacetic 
acid interferes with the test, as do also, to some extent, bile 
and albumin. 

Ruhemann's reagent consists of iodin and potassium iodid 
each, 1.5 parts; absolute alcohol, 15 parts; and distilled water, 
185 parts. 

6, Ammonia. — A small amount of ammonia, com- 
bined with hydrochloric, phosphoric, and sulphuric 
acids is always present. Estimated as NH3, the normal 
average is about 0.7 gm. in twenty-four hours. This 
represents 4 to 5 per cent, of the total nitrogen of the 
urine, ammonia standing next to urea in this respect. 

Under ordinary conditions, most of the ammonia 
which results from the metabolic processes is trans- 
formed into urea. When, however, acids are present 
in excess, either from ingestion of mineral acids or 
7 



98 THE URINE 

from abnormal production of acids within the body 
(as in fevers, diabetes, pernicious vomiting of preg- 
nancy, etc.), ammonia combines with them and is so 
excreted, urea being correspondingly decreased. It is 
thus that the body protects itself against acid intoxica- 
tion, A marked increase of ammonia is, therefore, im- 
portant chiefly as an index of the tendency to acidosis, 
particularly that associated with the presence of di- 
acetic and oxybutyric acids. 

In diabetes mellitus ammonia elimination may reach 
4 or 5 gm. daily. It is likewise markedly increased in 
pernicious vomiting of pregnancy, but not in nervous 
vomiting; and in conditions in which the power to syn- 
thesize urea is interfered with, notably cirrhosis and 
other destructive diseases of the liver and conditions 
associated with deficient oxygenation. 

Quantitative Estimation. — The urine must be fresh, 
since decomposition increases the amount of ammonia. 
The following method is satisfactory for clinical pur- 
poses, though subject to some inaccuracies. 

Ronchese-Malfatti Formalin Test. — This depends upon 
the fact that when formalin is added to the urine, the am- 
monia combines with it, forming hexamethylene-tetramin. 
The acids with which the ammonia was combined are set 
free, and their quantity, ascertained by titration with sodium 
hydroxid, indicates the amount of ammonia. 

Take 10 c.c. of the urine in a beaker or evaporating dish, 
add 50 c.c. water and 10 drops of 0.5 per cent, alcoholic solu- 
tion of phenolphthaleln. Neutralize by adding a weak 
caustic soda or sodium carbonate solution until a permanent 
pink color appears. To 5 c.c. formalin add 15 c.c. water 
and neutralize in the same way. Pour the formalin into the 



CHEMIC EXAMINATION 99 

urine. The pink color at once disappears, owing to libera- 
tion of acids. Now add decinormal sodium hydroxid solution 
from a buret until the pink color just returns. Each cubic 
centimeter of the decinormal solution used in this titration 
corresponds to 0.0017 E^- of NH3. This must be multi- 
plied by ten to obtain the percentage from which the twenty- 
four-hour elimination of ammonia is calculated. 

The method is more complicated, but distinctly more 
accurate when carried out as suggested by E. W. Brown. 
Treat 60 c.c. of urine with 3 gm. of basic lead acetate, stir 
well, let stand a few minutes, and filter. Treat the filtrate 
with 2 gm. neutral potassium oxalate, stir well, and filter. 
Take 10 c.c. of the filtrate, add 50 c.c. water and 15 gm. 
neutral potassium oxalate, and proceed with the ammonia 
estimation as above outlined. 

B. Abnormal Constituents 

Those substances which appear in the urine only in 
pathologic conditions are of much more interest to the 
clinician than are those which have just been discussed. 
Among them are: proteins, sugars, the acetone bodies, 
bile, hemoglobin, and the diazo substances. The " pan- 
creatic reaction " and detection of drugs in the urine will 
also be discussed under this head. 

I. Proteins. — Of the proteins which may appear 
in the urine, serum-albumin and serum-globulin are the 
most important. Mucin, proteose, and a few others are 
found occasionally, but are of less interest. 

(i) Serum-albumin and Serum-globulin. — These two 
proteins constitute the so-called " urinary albumin." 
They usually occur together, have practically the same 
significance, and both respond to all the ordinary tests 
for " albumin." 



lOO THE URINE 

Their presence, or albuminuria, is probably the most 
important pathologic condition of the urine. It is 
either accidental or renal. The physician can make no 
greater mistake than to regard all cases of albuminuria 
as indicating kidney disease. 

Accidental or Jalse albuminuria is due to admixture 
with the urine of albuminous fluids, such as pus, blood, 
and vaginal discharge. The microscope will usually 
reveal its nature. It occurs most frequently in pyelitis, 
cystitis, and chronic vaginitis. 

Renal albuminuria refers to albumin which has passed 
from the blood into the urine through the walls of the 
kidney tubules or the glomeruli. 

Albuminuria sufficient to be recognized by clinical 
methods probably never occurs as a physiologic condi- 
tion, the so-called physiologic albuminuria appearing 
only under conditions which must be regarded as 
abnormal. Among these may be mentioned excessive 
muscular exertion in those unaccustomed to it; exces- 
sive ingestion of proteins; prolonged cold baths; and 
childbirth. In these conditions the albuminuria is 
slight and transient. 

There are certain other forms of albuminuria which 
have still less claim to be called physiologic, but which 
are not always regarded as pathologic. Among these 
are cyclic albuminuria, which regularly recurs at a 
certain period of the day, and orthostatic or postural 
albuminuria, which appears only when the patient is 
standing. They are rare and of obscure origin, and 
occur for the most part in neurasthenic subjects during 
adolescence. It is noteworthy in this connection that 
nephritis sometimes begins with a cycUc albuminuria. 



CHEMIC EXAMINATION f-fRpApwlOr 

""'^^f I rrr- * ^^ 

In pathologic conditions and in most, at least/ of'liE^ r/I "j 
"functional" conditions just enumerated, renal D^f. n f 
buminuria may be referred to one or more of the follow- 
ing causes. In nearly all cases it is accompanied by 
tube-casts. 

(a) Changes in the blood which render its albumin 
more diffusible, as in severe anemias, purpura, and 
scurvy. Here the albumin is small in amount. 

(Z>) Changes in circulation in the kidney, either anemia 
or congestion, as in excessive exercise, chronic heart 
disease, and pressure upon the renal veins. The quan- 
tity of albumin is usually, but not always, small. Its 
presence is constant or temporary, according to the 
cause. Most of the causes, if continued, will produce 
organic changes in the kidney. 

(c) Organic Changes in the Kidney. — These include 
the inflammatory and degenerative changes commonly 
grouped together under the name of nephritis, and also 
renal tuberculosis, neoplasms, and cloudy swelling due 
to irritation of toxins and drugs. The amount of al- 
bumin eliminated in these conditions varies from minute 
traces to 20 gm., or even more, in the twenty-four hours, 
and, except in acute processes, bears little relation to 
the severity of the disease. In acute and chronic 
parenchymatous nephritis the quantity is usually very 
large. In chronic interstitial nephritis it is small — 
frequently no more than a trace. It is small in cloudy 
swelling from toxins and drugs, and variable in renal 
tuberculosis and neoplasms. In amyloid disease of the 
kidney the quantity is usually small, and serum-globulin 
may be present in especially large proportion, or even 
alone. Roughly distinctive of serum-globulin is the 



I02 THE URINE 

appearance of an opalescent cloud when a few drops of 
the urine are dropped into a glass of distilled water. 

Detection of albumin depends upon its precipitation 
by chemicals or coagulation by heat. There are many 
tests, but none is entirely satisfactory, because other 
substances as well as albumin are precipitated. The 
most common source of error is mucin. The tests given 
here are widely used and can be recommended. They 
make no distinction between serum-albumin and serum- 
globulin. They are given as nearly as possible in order 
of their delicacy. Usually the best time to detect 
albumin is in the evening or a few hours after a meal. 

// is very important that urine to he tested for albumin 
he rendered clear hy filtration or centrifugation. This 
is too often neglected in routine work. When ordinary 
methods do not suffice, it can usually be cleared by 
shaking up with a little purified talc or animal charcoal 
and filtering. 

(i) Trichloracetic Acid Test. — The reagent consists of a 
saturated aqueous solution of trichloracetic acid to which 
magnesium sulphate is added to saturation. A simple 
saturated solution of the acid may be used, but addition of 
magnesium sulphate favors precipitation of globulin, and, 
by raising the specific gravity, makes the test easier to 
apply. 

Take a few cubic centimeters of the reagent in a test-tube 
or conical test glass, hold the tube or glass in an inclined 
position, and run the urine gently in by means of a pipet, so 
that it will form a layer on top of the reagent without mix- 
ing with it. If albumin be present, a white, cloudy ring will 
appear where the two fluids come in contact. The ring can 
be seen most clearly if viewed against a black background, 



CHEMIC EXAMINATION I03 

and one side of the tube or conical glass may be painted black 
for this purpose. 

This is an extremely sensitive test, but, unfortunately, 
both mucin and proteoses respond to it; urates, when abund- 
ant, may give a confusing white ring, and the reagent is com- 
paratively expensive. It is not much used in routine work 
except as a control to the less sensitive tests. 




Fig. 26. — Horismascope: adding the reagent. 

A most convenient instrument for applying this or any of 
the contact tests is sold under the name of " horismascope " 
(Fig. 26). 

(2) Robert's Test. — ^The reagent consists of pure nitric 
acid, I part, and saturated aqueous solution of magnesium 
sulphate, 5 parts. It is applied in the same way as the pre- 
ceding test. 

Albumin gives a white ring, which varies in density with 



I04 THE URINE 

the amount present. A similar white ring may be produced 
by primary proteose and resinous drugs. White rings or 
cloudiness in the urine above the zone of contact may result 
from excess of urates or mucus. Colored rings near the junc- 
tion of the fluids may be produced by urinary pigments, 
bile, or indicari. 

Robert's test is one of the best for routine work, although 
the various rings are apt to be confusing to the inexperienced. 
It is more sensitive than Heller's test, of which it is a modifica- 
tion, and has the additional advantage that the reagent is 
not so corrosive. 

(3) Purdy's Heat Test. — Take a test-tube two-thirds full 
of urine, add about one-sixth its volume of saturated solution 
of sodium chlorid, and 5 to 10 drops of 50 percent, acetic acid. 
Mix, and boil the upper inch. A white cloud in the heated 
portion shows the presence of albumin. 

This is a valuable test for routine work. It is simple, 
sufficiently accurate for clinical purposes, and has practically 
no fallacies. Addition of the salt solution, by raising the 
specific gravity, prevents precipitation of mucin. Proteose 
may produce a white cloud, which disappears upon boiling 
and reappears upon cooling. 

(4) Heat and Nitric Acid Test. — This is one of the oldest 
of the albumin tests, and if properly carried out, one of the 
best. Boil a small quantity of filtered urine in a test-tube and 
add about one-twentieth its volume of concentrated nitric 
acid. A white cloud or flocculent precipitate (which usually 
appears during the boiling, but if the quantity be very small 
only after addition of the acid) denotes the presence of albu- 
min. A similar white precipitate, which disappears upon 
addition of the acid, is due to earthy phosphates. The acid 
should not be added before boiling, and the proper amount 
should always be used; otherwise, part of the albumin may 
fail to be precipitated or may be redissolved. 



CHEMIC EXAMINATION 



105 



Quantitative Estimation. — The gravimetric, which is 
the most reliable method, is too elaborate for clinical 
work. Both Esbach's, which is very widely used, and 
the centrifugal method give fair results, but Tsuchiya's 
recent modification of the Esbach 
method is preferable to either. 

(i) Esbach's Method. — ^The urine must 
be clear, of acid reaction, and not con- 
centrated. Always filter before testing, 
and, if necessary, add acetic acid and dilute 
with water. Esbach's tube (Fig. 27) is es- 
sentially a test-tube with a mark U near 
the middle, a mark R near the top, and 
graduations J, i, 2, 3, etc., near the bot- 
tom. Fill the tube to the mark U with 
urine and to the mark R with the reagent. 
Close with a rubber stopper, invert slowly 
several times, and set aside in a cool place. 
At the end of twenty-four hours read off 
the height of the precipitate. This gives 
the amount of albumin in grams per liter, 
and must be divided by 10 to obtain the 
percentage. 

Esbach's reagent consists of picric acid, i 
2 gm., and distilled water, to make 100 c.c. 

(2) Tsuchiya's Method. — ^This is carried out in the same 
manner as the Esbach method, using the following reagent: 

Phosphotungstic acid 1.5 gm. 

96 per cent, alcohol 95.0 c.c. 

Concentrated hydrochloric acid 5-o " 

The urine should be diluted to a specific gravity not exceeding 
1.008. The method is said to be much more accurate than 



Fig. 27. — Esbach's 
albuminometer, im- 
proved form. 

gm., citric acid, 



I06 THE URINE 

the original Esbach method, particularly with small quanti- 
ties of albumin. 

(3) Purdy's Centrifugal Method. — This is detailed in 
the table on opposite page. The percentage by weight is 
approximately one-fiftieth of the bulk percentage. 

(2) Mucin. — Traces of the substances (mucin, mu- 
coid, etc.) which are loosely classed under this name are 
present in normal urine; increased amounts are observed 
in irritations and inflammations of the mucous mem- 
brane of the urinary tract. They are of interest chiefly 
because they may be mistaken for albumin in most of 
the tests. If the urine be diluted with water and acidi- 
fied with acetic acid, the appearance of a w'hite cloud in- 
dicates the presence of mucin. 

True mucin is a glyco-protein, and upon boiling with 
an acid or alkali, as in Fchling's test, yields a carbohy- 
drate substance which reduces copper. 

(3) Proteoses. — These are intermediate products in 
the digestion of proteins and are frequently, although 
incorrectly, called albumoses. Tw'O groups are generally 
recognized: primary proteoses, w^hich are precipitated 
upon half-saturation of their solutions wdth ammonium 
sulphate; and secondary proteoses, which are precipitated 
only upon complete saturation. 

The secondary proteoses have been observed in the 
urine in febrile and malignant diseases and chronic sup- 
purations, during resolution of pneumonia, and in many 
other conditions, but their clinical significance is in- 
definite. In pregnancy, albumosuria may be due to 
absorption of amniotic fluid. 

Primary proteoses are rarely encountered in the urine. 



CHEMIC EXAMINATION 



107 



PURDY'S QUANTITATIVE METHOD FOR ALBUMIN IN 
URINE (CENTRIFUGAL). 

Table showing the relation between the volumetric and gravimetric percentage 

of albumin obtained by means of the centrifuge with radius of six 

and three-quarter inches ; rate of speed, i$oo revolutions 

per minute ; time, three minutes. 



> 


> 




> 


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0.281 


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3iJ4 


0.656 


3-15 


Vt 


O.OI 


0.05 


14 


0.292 


1.4 


32 


0.667 


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0.016 


0.075 


14^ 


0.302 


145 


32^ 


0.677 


3-25 


I 


0.021 


0.1 


^5 , 


0-313 


1-5 


33 


0.687 


3-3 


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0.026 


0.125 


15^ 


0.323 


1-55 


3354 


0.698 


3-35 


IM 


0.031 


0.15 


16 


0-333 


1.6 


34 


0.708 


3-4 


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0.036 


0.175 


16^ 


0.344 


1.65 


3454 


0.719 


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2 


0.042 


0.2 


17 


0.354 


1-7 


35 


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0.047 


0.225 


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1-75 


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0.275 


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19 


0.396 


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0.325 


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0.406 


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3-75 


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0.875 


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654 


0.135 


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4254 


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28 


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Test. — Three cubic centimeters of 10 per cent, solution of ferrocyanid of 
potassium and 2 cubic centimeters of 50 per cent, acetic acid are added to 10 cubic 
centimeters of the urine in the percentage tube and stood aside for ten minutes, 
then placed in the centrifuge and revolved at rate of speed and time as stated at 
head of the table. If albumin is excessive, dilute the urine with water until 
volume of albumin falls below 10 per cent. Multiply result by the number 
of dilutions employed before using the table. 



Io8 THE URINE 

The protein known as the " Bence- Jones body " was 
originally classed under this head, but its true nature is 
uncertain. It is regarded as practically pathognomonic 
of multiple myeloma. 

The proteoses are not coagulable by heat, but are precipi- 
tated by such substances as trichloracetic acid and phos- 
photungstic acid. The primary proteoses, alone, are pre- 
cipitated by nitric acid. 

Proteoses may be detected by acidifying the urine with 
acetic acid, boiling and filtering while hot to remove mucin, 
albumin, and globulin, and testing the filtrate by the tri- 
chloracetic acid test. As above indicated, the nitric acid 
test, and half and complete saturation with ammonium sul- 
phate will separate the two groups. 

To detect Bence- Jones' body the urine is acidified with 
acetic acid and gently heated. If this substance be present, 
a precipitate will form at about 60° C. As the boiling-point 
is reached, it wholly or partially dissolves. It reappears 
upon cooling. 

2. Sugars. — Various sugars may at times be found in 
the urine. Dextrose is by far the most common, and is 
the only one of clinical importance. Levulose, lactose, 
and some others are occasionally met with. 

(i) Dextrose (Glucose). — It is probable that traces of 
glucose, too small to respond to the ordinary tests, are 
present in the urine in health. Its presence in appre- 
ciable amount constitutes " glycosuria." 

Transitory glycosuria is unimportant, and may occur 
in many conditions, as after general anesthesia and 
administration of certain drugs, in pregnancy, and 
following shock and head injuries. It may also occur 



CHEMIC EXAMINATION I09 

after eating excessive amounts of carbohydrates (ali- 
mentary glycosuria). 

Persistent glycosuria has been noted in brain injuries 
involving the floor of the fourth ventricle. As a rule, 
however, persistent glycosuria is diagnostic of diabetes 
mellitus, of which disease it is the essential symptom. 
The amount of glucose eliminated in diabetes is usually 
considerable, and is sometimes very large, reaching 500 
gm., or even more, in twenty-four hours, but it does not 
bear any uniform relation to the severity of the disease. 
Glucose may, on the other hand, be almost or entirely 
absent temporarily. 

Detection of Dextrose. — If albumin be present in more 
than traces, it must be removed by boiling and filtering. 

(i) Haines* Test. — Take about i dram of Haines' solution 
in a test-tube, boil, and add 6 or 8 drops of urine. A heavy 
yellow or red precipitate, which settles readily to the bottom, 
shows the presence of sugar. Neither precipitation of phos- 
phates as a light, flocculent sediment nor simple decolorization 
of the reagent should be mistaken for a positive reaction. 

This is one of the best of the copper tests, all of which 
depend upon the fact that in strongly alkaline solutions glucose 
reduces cupric hydrate to cuprous hydrate (yellow) or cup- 
rous oxid (red). They are somewhat inaccurate, because 
they make no distinction between glucose and less common 
forms of sugar; because certain normal substances, when 
present in excess, especially mucin, uric acid, and creatinin, 
may reduce copper, and because many drugs — e. g., chloral, 
-chloroform, copaiba, acetanilid, benzoic acid, morphin, 
sulphonal, salicylates — are eliminated as copper-reducing 
substances. To minimize these fallacies dilute the urine, if 
it be concentrated; do not add more than the specified amount 
of urine, and do not boil after the urine is added. 



no 



THE URINE 



Haines'' solution is prepared as follows: completely dissolve 
30 gr. pure copper sulphate in \ oz. distilled water, and add 
2 oz. pure glycerin; mix thoroughly, and add 5 oz. liquor 
potassae. The solution keeps well. 

(2) Fehling's Test. — Two solutions are required — one 
containing 34.64 gm. pure crystalline copper sulphate in 
500 c.c. distilled water; the other, 173 gm. Rochelle salt and 
100 gm. potassium hydroxid in 500' c.c. distilled water. Mix 
equal parts of the two solutions in a test-tube, dilute with 




Fig. 28. — Crystals of phenylclucosazone from diabetic urine — Kowarsky's test ( X 500). 

3 or 4 volumes of water, and boil. Add the urine a little at a 
time, heating, but not boiling, between additions. In the 
presence of glucose a heavy red or yellow precipitate will 
appear. The quantity of urine should not exceed that of the 
reagent. 

(3) Benedict's Test. — This new test promises to displace 
all other reduction tests for glucose. The reagent is said to 
be ten times as sensitive as Haines' or Fehling's, and not to 
be reduced by uric acid, creatinin, chloroform, or the alde- 
hyds. It consists of: 



CHEMIC EXAMINATION III 



17-3 gm. 


173-0 " 


200.0 " 


lOOO.O c.c. 



Copper sulphate (pure crystallized), 
Sodium or potassium citrate, 
Sodium carbonate (crystallized), 

(or IOC gm. of the anhydrous salt). 
Distilled water, to make 



Dissolve the citrate and carbonate in 700 c.c. of water, with 
the aid of heat, and filter. Dissolve the copper in 100 c.c. 
of water and pour slowly into the first solution, stirring con- 
stantly. Cool, and make up to one liter. The reagent keeps 
indefinitely. 

Take about 5 c.c. of this reagent in a test-tube, and add 
8 or 10 drops {not more) of the urine. Heat to vigorous 
boiling, keep at this temperature for one or two minutes, 
and allow to cool slowly. In the presence of glucose the 
entire body of the solution will be filled with a precipitate, 
which may be red, yellow, or green in color. When traces 
only of glucose are present, the precipitate may appear only, 
upon cooling. In the absence of glucose, the solution re- 
mains clear or shows only a faint, bluish precipitate, due to 
urates. 

(4) Phenylhydrazin Test. — Kowarsky^s Method. — In a wide 
test-tube take 5 drops pure phenylhydrazin, 10 drops glacial 
acetic acid, and i c.c. saturated solution of sodium chlorid. 
A curdy mass results. Add 2 or 3 c.c. urine, boil for at least 
two minutes, and set aside to cool. Examine the sediment 
with the microscope, using a two-thirds objective. If glucose 
be present, characteristic crystals of phenylglucosazone will 
be seen. These are yellow, needle-like crystals arranged 
mostly in clusters or in sheaves (Fig. 28). When traces only 
of glucose are present, the crystals may not appear for one- 
half hour or more. Best crystals are obtained when the fluid 
is cooled very slowly. It must not be agitated during 
cooling. 



112 THE URINE 

This is an excellent test for clinical work. It requires 
slightly more time than Haines' test, but more than compen- 
sates for this by increased accuracy. It is fully as sensitive 
as Haines', and has practically no fallacies excepting levulose, 
which is a fallacy for all tests but the polariscope. Other 
carbohydrates which are capable of forming crystals with 
phenylhydrazin are extremely imlikely to do so when the test 
is appUed directly to the urine by the method just detailed. 
Even if not used routinely, this test should always be resorted 
to when Haines' test gives a positive reaction in doubtful 
cases. 

Quantitative Estimation. — In quantitative work Feh- 
ling's solution, for so many years the standard, has been 
largely displaced by Purdy's, which avoids the heavy 
precipitate that so greatly obscures the end-reaction in 
Fehling's method. The older method is still preferred 
by many, and both are, therefore, given. The new 
method of Benedict is likewise included, since it appears 
to be more exact than any other titration method 
available for sugar work. Should the urine contain 
much glucose, it must be diluted before making 
any quantitative test, allowance being made for the 
dilution in the subsequent calculation. Albumin, if 
present, must be removed by acidifying a considerable 
quantity of urine with acetic acid, boiling, and filtering. 
The precipitate should then be washed with water and 
the washings added to the urine to bring it to its original 
volume. 

(i) Purdy's Method. — Take exactly 35 c.c. of Purdy's 
solution in a flask or beaker, add twice its volume of distilled 
water, heat to boiling, and, still keeping the solution hot, add 



CHEMIC EXAMINATION II3 

the urine very slowly from a buret until the blue color entirely 
disappears. Read off the amount of urine added; considering 
the strength of Purdy's solution, it is readily seen that this 
amount of urine contains 0.02 gm. of glucose, from which the 
amount in the twenty-four-hour urine, or the percentage, can 
easily be calculated. Example: Suppose that 2.5 c.c. of 
urine discharged the blue color of 35 c.c. of Purdy's solution. 
This amount of urine, therefore, contains exactly 0.02 gm. 
glucose, and the percentage is obtained from the equation: 
2.5 : 100 : : 0.02 : x, and x equals 0.8 per cent. If, then, 
the twenty-four-hour quantity of urine were 3000 c.c, the 
twenty-four-hour elimination of glucose would be found as 
follows: 100 : 3000 : : 0.8 : x, and x equals 24 gm. 

It will be found that after the test is completed the blue 
color slowly returns. This is due to reoxidation, and should 
not be mistaken for incomplete reduction. 

A somewhat simpler application of this method, which is 
accurate enough for clinical purposes, is as follows: Take 
84 c.c. (roughly, 9 c.c.) of Purdy's solution in a large test- 
tube, dilute with an equal volume of water, heat to boiling, 
and, while keeping the solution hot but not boUing, add the 
urine drop by drop from a medicine-dropper until the blue 
color is entirely gone. Toward the end add the drops very 
slowly, not more than 4 or 5 a minute. Divide 10 by the 
number of drops required to discharge the blue color; the 
quotient will be the percentage of glucose. If 20 drops were 
required, the percentage would be 10-^-20 = 0.5 P^^ cent. 
It is imperative that the drops be of such size that 20 of them 
will make i c.c. Test the dropper with urine, not water. If 
the drops are too large, draw out the tip of the dropper; if 
too small, file off the tip. 

Ptirdy's solution consists of pure crystalline copper sulphate, 
4.752 gm. ; potassium hydroxid, 23.5 gm.; ammonia (U. S. P.; 
sp. gr., 0.9), 350 c.c. ; glycerin, 7,8 c.c. ; distilled water, to make 
8 



114 



THE URINE 



looo c.c. Dissolve the copper sulphate and glycerin in 200 
c.c. of the water by aid of gentle heat. In another 200 c.c. 
pf water dissolve the potassium hydroxid. Mix the two solu- 
tions, and when cool, add the ammonia. Lastly, bring the 
whole up to 1000 c.c. with distilled water. This solution is 
pf such strength that the copper in 35 c.c. will be reduced by 
exactly 0.02 gm. of glucose. 

(2) Fehling's Method. — Take 10 c.c. Fehling's solution 
(made by mixing 5 c.c. each of the copper and alkaline solu- 
tions described on page no) in a flask or beaker, add three or 




Fig. 29. — Einhorn's saccharimeter. 



four volumes of water, boil, and add the urine very slowly from 
a buret until the solution is completely decolorized, heating 
but not boiling after each addition. 

The chief objection to Fehling's method is the difficulty 
of determining the end-point. The use of an " outside indi- 
cator," however, obviates this. When reduction is thought 
to be complete, a few drops of the solution are filtered through 
a fine-grained filter-paper on to a porcelain plate, quickly 
acidified with acetic acid, and mixed with a drop of 10 per 



CHEMIC EXAMINATION II5 

cent, potassium ferrocyanid. Immediate appearance of a 
red-brown color shows the presence of unreduced copper. 

Fehling's solution is of such strength that the copper in 
10 c.c. will be reduced by exactly 0.05 gm. of glucose. There- 
fore, the amount of urine required to decolorize the test solu- 
tion contains just 0.05 gm. glucose, and the percentage is 
easily calculated. 

(3) Benedict's Method. — The following modification of 
his copper solution has recently been offered by Benedict 
for quantitative estimations. 

The reagent consists of: 

Copper sulphate (pure crystallized), 18.0 gm. 

Sodium carbonate (crystallized), 200.0 " 

(or 100 gm. of the anhydrous salt). 

Sodium or potassium citrate, 200.0 " 

Potassium sulphocyanate, 125.0 '* 
5 per cent, potassium ferrocyanid solution, 5.0 c.c. 

Distilled water, to make looo.o " 

With the aid of heat dissolve the carbonate, citrate, and 
sulphocyanate in about 800 c.c. of the water and filter. 
Dissolve the copper in 100 c.c. of water and pour slowly 
into the other fluid, stirring constantly. Add the ferro- 
cyanid solution, cool, and dilute to 1000 c.c. Only the 
copper need be accurately weighed. This solution is of such 
strength that 25 c.c. are reduced by 0.05 gram glucose. It 
keeps well. 

To make a sugar estimation, take 25 c.c. of the reagent in 
a porcelain evaporating dish, add 10 to 20 grams sodium 
.carbonate crystals (or one-half this weight of the anhydrous 
salt) and a small quantity of powdered pumice-stone or tal- 
cum. Heat to boiling, and add the urine rather rapidly 
from a buret until a chalk-white precipitate forms and 
the blue color of the reagent begins to fade. After this 



Il6 THE URINE 

point is reached, add the urine a few drops at a time until 
the last trace of blue just disappears. This end-point is 
easily recognized. During the whole of the titration the 
mixture must be kept vigorously boiling. Loss by evap- 
oration must be made up by adding water. The quantity 
of urine required to discharge the blue color contains exactly 
0.05 gram glucoSe, and the percentage contained in the 
original sample is easily calculated. 

(4) Fermentation Method.— This is convenient and satis- 
factory, its chief disadvantage begin the time required. It de- 
pends upon the fact that glucose is fermented by yeast with 
evolution of CO2. The amount of gas evolved is an index of 
the amount of glucose. Einhorn's saccharimeter (Fig. 29) is 
the simplest apparatus. 

The urine must be so diluted as to contain not more than 
I per cent, of glucose. A fragment of fresh yeast cake about 
the size of a split-pea is mixed with a definite quantity of the 
urine measured in the tube which accompanies the appa- 
ratus. It should form an emulsion free from lumps or air- 
bubbles. The long arm of the apparatus is then filled with 
the mixture. At the end of fifteen to twenty-four hours fer- 
mentation will be complete, and the percentage of glucose can 
be read off upon the side of the tube. The result must then 
be multiplied by the degree of dilution. Since yeast itself 
sometimes gives ofif gas, a control test must be carried out 
with normal urine and the amount of gas evolved must be 
subtracted from that of the test. A control should also be 
made with a known glucose solution to make sure that the 
yeast is active. 

(5) Robert's Differential Density Method. — While this 
method gives only approximate results, it is convenient, and 
requires no special apparatus but an accurate urinometer. 
Mix a quarter of a yeast-cake with about 4 oz. of urine. 
Take the specific gravity and record it. Set the urine in a 
warm place for twenty-four hours or until fermentation is com- 



CHEMIC EXAMINATION II 7 

plete. Then cool to the temperature at which the specific 
gravity was originally taken, and take it again. The differ- 
ence between the two readings gives the number of grains 
of sugar per ounce, and this, multiplied by 0.234, gives the 
percentage of sugar. If the original reading is 1.035, ^^^ that 
after fermentation is 1.020, the urine contains 1.035 — 1.020 
= 15 grains of sugar per fluidounce; and the percentage equals 
15X0.234 = 3.5. 

(2) Levulose, or fruit-sugar, is very rarely present in 
the urine except in association with glucose, and has 
about the same significance. Its name is derived from 
the fact that it rotates polarized light to the left. It be- 
haves the same as glucose with all the ordinary tests, 
and is not readily distinguished except by polarization. 

(3) Lactose, or milk-sugar, is sometimes present in 
the- urine of nursing women and in that of women who 
have recently miscarried. It is of interest chiefly be- 
cause it may be mistaken for glucose. // reduces copper, 
hut does not ferment with yeast. In strong solution it can 
form crystals with phenylhydrazin, but is extremely 
unlikely to do so when the test is applied directly to the 
urine. 

(4) Pentoses. — These sugars are so named because 
they contain five atoms of oxygen. Vegetable giuns 
form their chief source. They reduce copper strongly 
but slowly, and give crystals with phenylhydrazin, but 
do not ferment with yeast. 

Pentosuria is uncommon. It has been noted after in- 
gestion of large quantities of pentose-rich substances, 
such as cherries, plums, and fruit-juices, and is said to 
be fairly constant in habitual use of morphin. It some- 
times accompanies glycosuria in diabetes. An obscure 



Il8 THE URINE 

chronic form of pentosuria without cHnical symptoms 
has been observed. 

Bial's Orcin Test. — Dextrose is first removed by fermen- 
tation. About 5 c.c. of Bial's reagent are heated in a test- 
tube, and after removing from the flame the urine is added 
drop by drop, not exceeding twenty drops in all. The ap- 
pearance of a green color denotes pentose. 

The reagent consists of: 

30 i)er cent, hydrochloric acid 500 c.c. 

10 ])er cent, ferric chlorid solution 25 drops 

Orcin i gram. 

3. Acetone Bodies.- -This is a group of closely related 
substances — acetone, diacetic acid, and beta-oxy butyric 
acid. Acetone is derived from decomposition of diacetic 
acid, and this in turn from beta-oxybutyric acid by oxida- 
tion. The origin of beta-oxybutyric acid is not definitely 
known, but it is probable that its chief, if not its only, 
source is in some obscure metabolic disturbance with 
abnormal destruction of fats. The three substances 
generally appear in the urine in the order mentioned. 
When the disturbance is mild, acetone only appears; as it 
becomes more marked, diacetic acid is added, and finally 
beta-oxybutyric acid appears. The {presence of beta- 
oxybutyric acid in the blood is probably the chief cause 
of the form of auto-intoxication known as " acid intoxi- 
cation." 

(i) Acetone. — Minute traces, too small for the ordi- 
nary tests, may be present in the urine under normal 
conditions. Larger amounts are not uncommon in 
fevers, gastro-intestinal disturbances, and certain ner- 
vous disorders. A notable degree of acetonuria has 



CHEMIC EXAMINATION II 9 

Kkewise been observed in pernicious vomiting of preg- 
nancy and in eclampsia. 

Acetonuria is practically always observed in acid 
intoxication, and, together with diaceturia, constitutes 
its most significant diagnostic sign. A similar or identi- 
cal toxic condition, always accompanied by acetonuria 
and often fatal, is now recognized as a not infrequent 
late effect of anesthesia, particularly of chloroform anes- 
thesia. This postanesthetic toxemia is more Hkely to 
appear, and is more severe when the urine contains any 
notable amount of acetone before operation, which sug- 
gests the importance of routine examination for acetone 
in surgical cases. 

Acetone is present in considerable amounts in many 
cases of diabetes mellitus, and is always present in severe 
cases. Its amount is a better indication of the severity 
of the disease than is the amount of sugar. A progres- 
sive increase is a grave prognostic sign. It can be 
diminished temporarily by more liberal allowance of 
carbohydrates in the diet. 

According to Folin, acetone is present in only small 
amounts in these conditions, the substance shown by 
the usual tests, particularly after distillation of the 
urine, being really diacetic acid. In this connection, 
Frommer's test is to be recommended, since it does not 
require distillation, and does not react to diacetic acid 
unless too great heat is applied. 

Detection of Acetone. — The urine may be tested di- 
rectly, but it is best to distil it after adding a little phos- 
phoric or hydrochloric acid to prevent foaming, and to 
test the first few cubic centimeters of distillate. A 
simple distilling apparatus is shown in Fig. 30. The 



I20 



THE URINE 



test-tube may be attached to the delivery tube by means 
of a two-hole rubber cork as shown, the second hole 
serving as air vent, or, what is much less satisfactory, 
it may be tied in place with a string. Should the vapor 
not condense well, the test-tube may be immersed in a 
glass of cold water. 




I " 




Fig. 30. — A simple distillins apparatus. 



When diacetic acid is present, a considerable pro- 
portion will be converted into acetone during distilla- 
tion. 



(i) Giuining's Test. — To a few cubic centimeters of urine 
or distillate in a test-tube add a few drops of tincture of iodin 
and of ammonia alternately until a heavy black cloud appears. 
This cloud will gradually clear up, and if acetone be present, 
iodoform, usually crystalline, will separate out. The iodoform 
can be recognized by its odor, especially upon heating (there 



CHEMIC EXAMINATION 121 

is danger of explosion if the mixture be heated before the 
black cloud disappears), or by detection of the crystals mi- 
croscopically. The latter only is safe, unless one has an 
imusually acute sense of smell. Iodoform crystals are yel- 
lowish, six-pointed stars or six-sided plates (Fig. 31), 

This modification of Lieben's test is less sensitive than the 
original, but is sufficient for all clinical work; it has the ad- 
vantage that alcohol does not cause confusion, and especially 
that the sediment of iodoform is practically always crystalline. 
When applied directly to the urine, phosphates are precipi- 










. ^^tCs^T — - 

L-. ., 

Fig. 31. — Iodoform crystals obtained in several tests for acetone by Gunning's method 
(X about 600). 

tated and may form star-shaped crystals which are very con- 
fusing to the inexperienced. Albumin prevents formation 
of the crystals, and when it is present, the urine must be dis- 
tilled for the test. 

(2) Lange's Test. — This is a modification of the well- 
known Legal test. It is more sensitive and gives a sharper 
end-reaction. To a small quantity of urine add about one- 
twentieth its volume (i drop for each i c.c.) of glacial acetic 
acid and a few drops of fresh concentrated aqueous solution of 
sodium nitroprussid, and gently run a little ammonia upon its 



122 THE URINE 

surface. If acetone be present, a purple ring will form within 
a few minutes at the junction of the two fluids. 

(3) Frommer's Test. — This test has proved very satis- 
factory in the hands of the writer. The urine need not be 
distilled. Alkalinize about 10 c.c. of the urine with 2 or 3 c.c. 
of 40 per cent, caustic soda solution, add 10 or 12 drops of 
10 per cent, alcoholic solution of salicylous acid (salicyl 
aldehyd), heat the upper portion to about 70° C. (it should 
not reach the boiling-point), and keep at this temperature 
five minutes or longer. In the presence of acetone an orange 
color, changing to deep red, appears in the heated portion. 

The test can be made more definite by adding the caustic 
soda in substance (about i gram), and before it goes into 
solution adding the salicyl aldehyd and warming the lower 
portion. 

(2) Diacetic acid occurs in the same conditions as 
acetone, but is less frequent and has more serious signifi- 
cance. In diabetes its presence is a grave symptom and 
often forewarns of approaching coma. It rarely or never 
occurs without acetone. 

Detection: — The urine must be fresh. 

(i) Gerhardt's Test. — To a few cubic centimeters of the 
urine add solution of ferric chlorid (about 10 per cent.) drop 
by drop until the phosphates are precipitated; filter and add 
more of the ferric chlorid. If diacetic acid be present, the 
urine will assume a Bordeaux-red color which disappears 
upon boiling. A red or violet color which does not disappear 
upon boiling may be produced by other substances, as phenol, 
salicylates, and antipyrin. 

(2) Lindemann's Test. — To about 10 c.c. of urine add 
5 drops 30 per cent, acetic acid, 5 drops Lugol's solution, and 
2 or 3 c.c. chloroform, and shake. The chloroform does not 
change color if diacetic acid be present, but becomes reddish 



CHEMIC EXAMINATION 1 23 

violet in its absence. This test is claimed by its advocates to 
be more sensitive and more reliable than Gerhardt's. 

(3) Oxybutyric acid has much the same significance 
as diacetic acid, but is of more serious import. There is 
no satisfactory clinical test for it. 

4. Bile. — Bile appears in the urine in all diseases which 
produce jaundice, often some days before the skin be- 
comes yellow; and in many disorders of the liver not 
severe enough to cause jaundice. It also occurs in dis- 
eases with extensive and rapid destruction of red blood- 
corpuscles. Both bile-pigment and bile acids may be 
found. They generally occur together, but the pigment 
is not infrequently present alone. Bilirubin, only, oc- 
curs in freshly voided urine, the other pigments (bili- 
verdin, bilifuscin, etc.) being produced from this by 
oxidation as the urine stands. The acids are almost 
never present without the pigments, and are, therefore, 
seldom tested for clinically. 

Detection of Bile-pigment. — Bile-pigment gives the 
urine a greenish-yellow, yellow, or brown color, which 
upon shaking is imparted to the foam. Cells, casts, and 
other structures in the sediment may be stained brown or 
yellow. This, however, should not be accepted as prov- 
ing the presence of bile without further tests. 

(i) Smith's Test. — Overlay the urine with tincture of iodin 
diluted with nine times its volume of alcohol. An emerald- 
green ring at the zone of contact shows the presence of bile- 
pigments. It is convenient to use a conical test-glass, one 
side of which is painted white. 

(2) Gmelin's Test. — This consists in bringing shghtly 
yellow nitric acid into contact with the urine. A play of 



124 THE URINE 

colors, of which green and violet are most distinctive, denotes 
the presence of bile-pigment. Colorless nitric acid will be- 
come yellow upon standing in the sunlight. The test may be 
applied in various ways : by overlaying the acid with the urine; 
by bringing a drop of each together upon a porcelain plate; 
by filtering the urine through thick filter-paper, and touching 
the paper with a drop of the acid; and, probably best of all, 
by precipitating with lime-water, filtering, and touching the 
precipitate with a drop of the acid. In the last method bili- 
rubin is carried down as an insoluble calcium compound. 

Detection of Bile Acids. — Hay's test is simple, sensi- 
tive, and fairly reliable, and will, therefore, appeal to 
the practitioner. It depends upon the fact that bile 
acids lower surface tension. Other tests require isola- 
tion of the acids for any degree of accuracy. 

Hay's Test. — Upon the surface of the urine, which must not 
be warm, sprinkle a little finely powdered sulphur. If it 
sinks at once, bile acids are present to the amount of o.oi 
per cent, or more; if only after gentle shaking, 0.0025 P^r 
cent, or more. If it remains floating, even after gentle 
shaking, bile acids are absent. 

5. Hemoglobin. — The presence in the urine of hemo- 
globin or pigments directly derived from it, accompanied 
by few, if any, red corpuscles, constitutes hemoglobinuria. 
It is a rare condition, and must be distinguished from 
hematuria, or Uood in the urine, which is common. In 
both conditions chemic tests will show hemoglobin, but 
in the latter the microscope will reveal the presence of 
red corpuscles. Urines which contain notable amounts 
of hemoglobin have a reddish or brown color, and may 
deposit a sediment of brown, granular pigment. 



CHEMIC EXAMINATION I25 

Hemoglobinuria occurs when there is such extensive 
destruction of red blood-cells within the body that the 
liver cannot transform all the hemoglobin set free into 
bile-pigment. The most important examples are seen in 
poisoning, as by mushrooms and potassimn chlorate, 
in scurvy and purpura, in malignant malaria (black water 
.fever), and in the obscure condition known as " paroxys- 
mal hemoglobinuria." This last is characterized by the 
appearance of large quantities of hemoglobin at inter- 
vals, usually following exposure to cold, the urine remain- 
ing free from hemoglobin between the attacks. 

Detection. — Teichmann's test (p. 274) may be applied 
to the precipitate after boiling and filtering, but the 
guaiac test is more convenient in routine work. 

Guaiac Test. — Mix equal parts of " ozonized " turpentine 
and fresh tincture of guaiac which has been diluted with 
alcohol to a light sherry- wine color. In a test-tube or conical 
glass overlay the urine with this mixture. A bright blue ring 
will appear at the zone of contact within a few minutes if 
hemoglobin be present. The guaiac should be kept in an 
amber-colored bottle. Fresh turpentine can be " ozonized " 
by allowing it to stand a few days in an open vessel in the 
sunlight. 

This test is very sensitive, and a negative result proves the 
absence of hemoglobin. Positive. results are not conclusive, 
because numerous other substances — few of them likely to be 
found in the urine — may produce the blue color. That most 
likely to cause confusion is pus, but the blue color produced 
by it disappears upon heating. The thin film of copper 
often left in a test-tube after testing for sugar may give the 
reaction, as may also the fumes from an open bottle of bromin. 



126 THE URINE 

6. Alkapton Bodies.- -The name, alkaptonuria, has 
been given to a condition in which the urine turns 
reddish-brown upon standing and strongly reduces 
copper (but not bismuth), owing to the presence of 
certain substances which result from imperfect protein 
metabolism. The change of color takes place quickly 
when fresh urine is alkalinized, hence the name, alkapton 
bodies. 

Alkaptonuria is unaccompanied by other symptoms, 
and has little clinical importance. Only about forty-five 
cases, mostly congenital, have been reported. The 
change in color of the urine and the reduction of copper 
with no reduction of bismuth nor fermentation with 
yeast would suggest the condition. 

7. Melanin. — Urine which contains melanin likewise 
darkens upon exposure to the air, assuming a dark 
brown or black color. This is due to the fact that the 
substance is eliminated as a chromogen — melanogen — 
which is later converted into the pigment. 

Melanuria occurs in most, but not all, cases of mela- 
notic sarcoma. Its diagnostic value is lessened by the 
fact that it has been observed in other wasting diseases. 

Tests for Melanin. — (i) Addition of ferric chlorid gives a 
gray precipitate which blackens on standing. 

(2) Bromin water causes a yellow precipitate which 
gradually turns black. 

8. Diazo Substances. — Certain unknown substances 
sonretimes present in the urine give a characteristic 
color reaction — the " diazo reaction " of Ehrlich — when 
treated with diazo-benzol-sulphonic acid and ammonia. 



CHEMIC EXAMINATION 1 27 

This reaction has much cUnical value, provided its limi- 
tations be recognized. It is at best an empirical test 
and must be interpreted in the light of cHnical symptoms. 
Although it has been met with in a considerable number 
of diseases, its usefulness is practically limited to ty- 
phoid fever, tuberculosis, and measles. 

(i) Tjrphoid Fever. — Practically all cases give a 
positive reaction, which varies in intensity with the 
severity of the disease. It is so constantly present that 
it is sometimes said to be " negatively pathognomonic ": 
if negative upon several successive days at a stage of the 
disease when it should be positive, typhoid is almost 
certainly absent. Upon the other hand, a reaction 
when the urine is highly diluted (i : 50 or more) has 
much positive diagnostic value, since this dilution pre- 
vents the reaction in most conditions which might be 
mistaken for typhoid; but it should be noted that mild 
cases of typhoid may not give it at this dilution. Ordi- 
narily the diazo appears a little earlier than the Widal 
reaction, — about the fourth or fifth day, — but it may be 
delayed. In contrast to the Widal, it begins to fade 
about the end of the second week, and soon thereafter 
entirely disappears. An early disappearance is a favor- 
able sign. It reappears during a relapse, and thus helps 
to distinguish between a relapse and a complication, in 
which it does not reappear. 

(2) Tuberculosis. — The diazo reaction has been ob- 
tained in many forms of the disease. It has little or 
no diagnostic value. Its continued presence in pul- 
monary tuberculosis is, however, a grave prognostic 
sign, even when the physical signs are slight. After it 
once appears it generally persists more or less intermit- 



128 THE URINE 

tently until death, the average length of life after its 
appearance being about six months. The reaction is 
often temporarily present in mild cases during febrile 
complications, and has then no significance. 

(3) Measles. — A positive reaction is usually obtained 
in measles, and may help to distinguish this disease 
from German measles, in which it does not occur. It 
generally appears before the eruption and remains about 
five days. 

Technic. — Although the test is really a very simple one, 
careful attention to technic is imperative. Many of the early 
workers were very lax in this regard. Faulty technic and 
failure to record the stage of the disease in which the tests 
were made have probably been responsible for the bulk of the 
conflicting results reported. 

Certain drugs often given in tuberculosis and typhoid 
interfere with or prevent the reaction. The chief are creosote, 
tannic acid and its compounds, opium and its alkaloids, salol, 
phenol, and the iodids. The reagents are: 

(i) Saturated solution sulphanilic acid in 5 per cent, 
hydrochloric acid. 

(2) 0.5 per cent, aqueous solution sodium nitrite. 

(3) Strong ammonia. 

Mix 100 parts of (i) and one part of (2). In a test-tube 
take equal parts of this mixture and the urine, and pour i or 
2 c.c. of the ammonia upon its surface. If the reaction be 
positive, a garnet ring will form at the junction of the two 
fluids; and upon shaking, a distinct pink color will be imparted 
to the foam. The color of the foam is the essential feature. 
If desired, the mixture may be well shaken before the ammonia 
is added: the pink color will then instantly appear in that 
portion of the foam which the ammonia has reached, and can 
be readily seen. The color varies from eosin-pink to deep 



CHEMIC EXAMINATION 1 29 

crimson, depending upon the intensity of the reaction. It is 
a pure pink or red; any trace of yellow or orange denotes a 
negative reaction. A doubtful reaction should be considered 
negative. 

9. Pancreatic Reaction.— Cammidge has shown that 
in cases of pancreatitis a substance capable of forming 
crystals with phenylhydrazin can be developed by boiling 
the urine with a mineral acid, and has offered the follow- 
ing test as an aid in diagnosis of this obscure condition. 
The nature both of this substance and the antecedent 
substance from which it is derived is not known. As 
originally proposed, the test was complicated and prob- 
ably not trustworthy, but with his improved and sim- 
plified technic, Cammidge has had very promising results. 
In 200 consecutive examinations in which the diagnosis 
was confirmed, postmortem or at operation, 67 cases of 
pancreatitis (65 chronic, 2 acute) gave positive reactions; 
4 cases of cancer of the pancreas were positive, 1 2 nega- 
tive; 4 cases in which no pancreatitis was found were 
positive, 113 were negative. Normal urines do not give 
the reaction. The difficulty and importance of diag- 
nosis in pancreatitis warrant inclusion of the method 
here, even though more recent work indicates that its 
value is not so great as originally claimed. 

While the test is somewhat tedious, all the manipula- 
tions are simple and require no apparatus but flasks, 
test-tubes, and funnels. 

Technic. — Careful attention to detail is imperative. An 

ordinary routine examination is first made. Albumin and 

sugar, if present, must be removed: the former, by acidifying 

with acetic acid, boiling, and filtering; the latter, by fermenta- 

9 



I30 



THE URINE 



tion with yeast after the first step of the method proper. An 
alkahne urine should be made sUghtly acid with hydrochloric 
acid. 

(i) Forty cubic centimeters of the urine, which has been 
rendered perfectly clear by repeated filtration through the 
same filter-paper are placed in a small flask, treated with i 
c.c. concentrated hydrochloric acid and gently boiled on a 




Fig. 32. — "Pancreatic rcacti^ 



1" ll,lsk■^ tilti'il with fuiiiu'l conilcnsers on a sand-bath 
(Rohson and Cammidge). 



sand-bath for ten minutes, a funnel wath long stem being 
placed in the neck of the flask to act as a condenser (Fig. 32). 
After boiling, the urine is cooled in a stream of cold water and 
brought to its original bulk with distilled w^ater; 8 gm. of 
lead carbonate are then added to neutralize the acid. The 
fluid is allowed to stand a few minutes and then filtered 
through well-moistened fine-grain filter-paper until perfectly 
clear. 



CHEMIC EXAMINATION 131 

(2) The filtrate is shaken up with 8 gm. powdered tribasic 
lead acetate and filtered. The excess of lead is then removed 
by passing hydrogen sulphid gas through the fluid (see page 
135) or by shaking well with 4 gm. finely powdered sodium 
sulphate, heating to boiling, cooling to as low a temperature 
as possible in a stream of water, and filtering as before until 
perfectly clear. 




Fig. 33. — Improved "pancreatic reaction." Crystals obtained from a case of chronic 
pancreatitis with gall-stones in the common duct (X200) (from a photo by P. J. Cam- 
midge). 

(3) Ten cubic centimeters of the filtrate are then made 
up to 17 c.c. with distilled water, and added to a mixture of 
0.8 gm. phenylhydrazin hydrochlorate, 2 gm. powdered so- 
dium acetate, and i c.c. 50 per cent, acetic acid in a small flask 
with funnel condenser. This is boiled on a sand-bath for ten 
minutes, and filtered while hot through filter-paper moistened 
with hot water into a test-tube with a 15 c.c. mark. Should 



132 



THE URINE 



the filtrate not reach this mark, make up to 15 c.c. with hot 
distilled water. Allow to cool slowly. 

(4) In well-marked cases of pancreatitis a yellow pre- 
cipitate appears within a few hours; in milder cases, it may not 
appear for twelve hours. The microscope shows this sediment 
to consist of " Ions;;, light yellow, flexible, hair-like crystals 
arranged in sheaves, which, when irrigated with 33 per cent. 
suljihuric acid, melt away and disappear in ten to fifteen sec- 
onds after the acid first touches them " (Fig. 33). 

(5) To exclude traces of glucose which might be overlooked 
in the preliminary examination a control test should be carried 
out in the same manner with omission of step i. 

10. Drugs.— The effect of various drugs upon the 
color of the urine has been mentioned (p. 71). Most 
poisons are eliminated in the urine, but their detection 
is more properly discussed in works upon toxicology. A 
few drugs which are of interest to the practitioner, and 
which can be detected by comparatively simple methods, 
are mentioned here. 

Acetanilid and Phenacetin. — The urine is evaporated 
by gentle heat to about half its volume, boiled for a few 
minutes with about one-fifth its volume of strong hydro- 
chloric acid, and shaken out with ether. The ether is 
evaporated, the residue dissolved in water, and the 
following test applied : To about 10 c.c. are added a few 
cubic centimeters of 3 per cent, phenol, followed by a 
weak solution of chromium trioxid (chromic acid) drop 
by drop. The fluid assumes a red color, which changes 
to blue when ammonia is added. If the urine is very 
pale, extraction with ether may be omitted. 

Antipjn-in. — This drug gives a dark-red color when a 
few drops of 10 per cent, ferric chlorid are added to the 



CHEMIC EXAMINATION 133 

urine. The color does not disappear upon boiling, which 
excludes diacetic acid. 

Arsenic. — Reinsch's Test. — Add to the urine in a test- 
tube or small flask about one-seventh its volume of hy- 
drochloric acid, introduce a piece of bright copper-foil 
about one-eighth inch square, and boil for several min- 
utes. If arsenic be present, a dark-gray film is deposited 
upon the copper. The test is more delicate if the urine 
be concentrated by slow evaporation. This test is well 
known and is widely used, but is not so reUable as the 
following. 

GutzeWs Test. — In a large test-tube place a little 
arsenic-free zinc, and add 5 to 10 c.c. pure dilute hydro- 
chloric acid and a few drops of iodin solution (Gram's 
solution will answer), then add 5 to 10 c.c. of the urine. 
At once cover the mouth of the tube with a filter-paper 
cap moistened with saturated aqueous solution of silver 
nitrate (i: i). If arsenic be present, the paper quickly 
becomes lemon-yellow, owing to formation of a com- 
pound of silver arsenid and silver nitrate, and turns black 
when touched with a drop of water. To make sure that 
the reagents are arsenic-free, the paper cap may be ap- 
plied for a few minutes before the urine is added. 

Atropin will cause dilatation of the pupil when a few 
drops of the urine are placed in the eye of a cat or rabbit. 

Bromids can be detected by acidifying about 10 c.c. of 
the urine with dilute sulphuric acid, adding a few drops 
of fuming nitric acid and a few cubic centimeters of 
chloroform, and shaking. In the presence of bromin the 
chloroform, which settles to the bottom, assumes a yellow 
color. 

Iodin from ingestion of iodids or absorption from 



134 THE URINE 

iodoform dressings is tested for in the same way as the 
bromids, the chloroform assuming a pink to reddish- 
violet color. To detect traces, a large quantity of urine 
should be rendered alkaline with sodium carbonate and 
great Iv concentrated by evaporation before testing. 

Lead. No simple method is sufficiently sensitive to 
detect the traces of lead which occur in the urine in 
chronic poisoning. Of the more sensitive methods, that 
of Arthur Lederer is probably best suited to the prac- 
titioner: 

It is essential that all apparatus used be lead-free. 
Five hundred cubic centimeters of the urine are acidified 
with 70 c.c. pure sulphuric acid, and heated in a beaker 
or porcelain dish. About 20 to 25 gm. of potassium 
persulphate are added a little at a time. This should 
decolorize the urine, leaving it only slightly yellow. If 
it darkens upon heating, a few more crystals of potassium 
persulphate are added, the burner being first removed to 
prevent boiling over ; if it becomes cloudy, a small amount 
of sulphuric acid is added. It is then boiled until it has 
evaporated to 250 c.c. or less. After cooling, an equal 
volume of alcohol is added, and the mixture allowed to 
stand in a cool place for four or five hours, during which 
time all the lead will be precipitated as insoluble sulphate. 

The mixture is then filtered through a small, close- 
grained filter-paper (preferably an ashless, quantitative 
filter-paper), and any sediment remaining in the beaker 
or dish is carefully washed out with alcohol and filtered. 
A test-tube is placed underneath the funnel; a hole is 
punched through the tip of_the filter with a small glass rod, 
and all the precipitate (which may be so slight as to be 
scarcely visible) washed down into the test-tube with a 



CHEMIC EXAMINATION 135 

jet of distilled water from a wash-bottle, using as little 
water as possible. Ten cubic centimeters will usually 
sufl&ce. This fluid is then heated, adding crystals of 
sodium acetate until it becomes perfectly clear. It now 
contains all the lead of the 5cxd c.c. urine in the form of 
lead acetate. It is allowed to cool, and hydrogen sulphid 
gas is passed through it for about five minutes. The 
slightest yellowish-brown discoloration indicates the pres- 
ence of lead. A very slight discoloration can be best seen 




Fig. 34. — A simple hydrogen sulphid generator. 

when looked at from above. For comparison, the gas 
may be passed through a test-tube containing an equal 
amount of distilled water. The quantity of lead can be 
determined by comparing the discoloration with that 
produced by passing the gas through lead acetate (sugar 
of lead) solutions of known strength. One gram of lead 
acetate crystals contains 0.54 gram of lead. Hydrogen 
sulphid is easily prepared in the simple apparatus shown 
in Fig. 34. A small quantity of iron sulphid is placed 



136 THE URINE 

in the test-tube; a little dilute hydrochloric acid is added; 
the cork is replaced; and the delivery tube is inserted to 
the bottom of the fluid to be tested. 

Mercury. — Traces can be detected in the urine for a 
considerable time after the use of mercury compounds 
by ingestion or inunction. 

About a liter of urine is acidified with 10 c.c. hydro- 
chloric acid, and a small piece of copper-foil or gauze is 
introduced. This is gently heated for an hour, and 
allowed to stand for twenty-four hours. The metal is 
then removed, and washed successively with very dilute 
sodium hydroxid solution, alcohol, and ether. When 
dry. it is placed in a long, slender test-tube, and the lower 
portion of the tube is heated to redness. If mercury be 
present, it will volatilize and condense in the upper por- 
tion of the tube as small, shining globules which can be 
seen with a hand-magnifier or low power of the micro- 
scope. If, now, a crystal of iodin be dropped into the 
tube and gently heated, the mercury upon the side of the 
tube is changed first to the yellow iodid, and later to the 
red iodid, which are recognized by their color. 

Morphin. — Add sufficient ammonia to the urine to 
render it distinctly ammoniacal, and shake thoroughly 
with a considerable quantity of pure acetic ether. Sepa- 
rate the ether and evaporate to dryness. To a httle of 
the residue in a watch-glass or porcelain dish add a few 
drops of formaldehyd-sulphuric acid, which has been 
freshly prepared by adding one drop of formalin to i c.c. 
pure concentrated sulphuric acid. If morphin be pres- 
ent, this will produce a purple-red color, which changes 
to violet, blue-violet, and finally nearly pure blue. 

Phenol. — As has been stated, the urine following 



CHEMIC EXAMINATION 137 

phenol poisoning turns olive-green and then brownish- 
black upon standing. Tests are of value in recognizing 
poisoning from ingestion and in detecting absorption 
from carbolized dressings. 

The urine is acidulated with hydrochloric acid and 
distilled. To the first few cubic centimeters of distillate 
is added lo per cent, solution of ferric chlorid drop by 
drop. The presence of phenol causes a deep amethyst- 
blue color, as in Uffelmann's test for lactic acid. 

Phenolphthalein, which is now widely used as a ca- 
thartic, gives a bright pink color when the urine is ren- 
dered alkaline with caustic soda. 

Quinin. — A considerable quantity of the urine is ren- 
dered alkaline with ammonia and extracted with ether; 
the ether is evaporated, and a portion of the residue dis- 
solved in about twenty drops of dilute alcohol. The 
alcoholic solution is acidulated with dilute sulphuric 
acid, a drop of an alcoholic solution of iodin (tincture 
of iodin diluted about ten times) is added, and the mix- 
ture, is warmed. Upon cooling, an iodin compound of 
quinin (herapathite) will separate out in the form of a 
microcrystalline sediment of green plates. 

The remainder of the residue may be dissolved in a 
little dilute sulphuric acid. This solution will show a 
characteristic blue fluorescence when quinin is present. 

Resinous drugs cause a white precipitate like that of 
albumin when strong nitric acid is added to the urine. 
This is dissolved by alcohol. 

Salicylates, salol, and similar drugs give a bluish- 
violet color, which disappears upon heating, upon addi- 
tion of a few drops of 10 per cent, ferric chlorid solution. 
When the quantity of salicylates is small, the urine may 



138 THE URINE 

be acidified with hydrochloric acid and extracted with 
ether, the ether evaporated, and the test applied to an 
aqueous solution of the residue. 

Tannin and its compounds appear in the urine as 
gallic acid, and the urine becomes greenish-black (inky, 
if much gallic acid be present) when treated with a solu- 
tion of ferric chlorid. 

III. MICROSCOPIC EXAMINATION 

A careful microscopic examination will often reveal 
structures of great diagnostic importance in urine which 
seems perfectly clear, and from which only very slight 
sediment can be obtained with the centrifuge. Upon the 
other hand, cloudy urines with abundant sediment are 
often shown by the microscope to contain nothing of 
clinical significance. 

Since the nature of the sediment soon changes, the 
urine must be examined while fresh, preferably within six 
hours after it is voided. The sediment is best obtained 
by means of the centrifuge. If a centrifuge is not 
available, the urine may be allowed to stand in a conical 
test-glass for six to twenty-four hours after adding some 
preservative (p. 69). The " torfuge " (Fig. 35) is said 
to be a very satisfactory substitute for the centrifuge, 
and is readily portable. 

A small amount of the sediment should be transferred 
to a slide by means of a pipet. It is very important to do 
this properly. The best pipet is a small glass tube which 
has been drawn out at one end to a tip with rather small 
opening. The tube or glass containing the sediment is 
held on a level with the eye. the larger end of the pipet is 
closed with the index-finger, which must be dry, and the 



MICROSCOPIC EXAMINATION 



139 



tip is carried down into the sediment. By carefully 
loosening the finger, but not entirely removing it, a small 
amount of the sediment is then allowed to run slowly into 
the pipet. Slightly rotating the pipet will aid in accom- 
plishing this, and at the same time will serve to loosen 
any structures which cling to the bottom of the tube. 
After wiping off the urine which adheres to the outside, 
a drop from the pipet is placed upon a clean slide. 
A hair is then placed in the drop, and a large cover-glass 




Fig. 35. — Wetherill's torfuge. 

applied. Many workers use no cover. This offers a 
thicker layer and larger area of urine, the chance of find- 
ing scanty structures being proportionately increased. It 
has the disadvantage that any jarring of the room (as by 
persons walking about) sets the microscopic field into 
vibratory motion and makes it impossible to see an3^thing 
clearly; and since it does not allow of the use of high- 
power objectives, one cannot examine details as one often 
wishes to do. A large cover-glass with a hair beneath it 
avoids these disadvantages, and gives enough urine to 
find any structures which are present in sufficient 



I40 THE URINE 

number to have clinical significance, provided other 
points in the technic have been right. It is best, how- 
ever, to examine several drops; and, when the sediment 
is abundant, drops from the upper and lower portions 
should be examined separately. 

In examining urinary sediments microscopically no 
fault is so common, nor so fatal to good results, as im- 
proper illumination (see Fig. 4), and none is so easily 
corrected. The light should be central and very sub- 
dued for ordinary work, but oblique illumination, ob- 
tained by swinging the mirror a little out of the optical 
axis, will be found helpful in identifying certain dehcate 
structures like hyaline casts. The 16 mm. objective 
should be used as a finder, while the 4 mm. is reserved 
for examining details. An experienced worker will rely 
almost wholly upon the lower power. 

It is well to emphasize that the most common errors 
which result in failure to find important structures, when 
present, are lack of care in transferring the sediment to the 
slide, too strong illumination, and too great magnification. 

In order to distinguish between similar structures it is 
often necessary to watch the effect upon them of certain 
reagents. This is especially true of the various unorgan- 
ized sediments. They very frequently cannot be identi- 
fied from their form alone. With the structures still in 
focus, a drop of the reagent may be placed at one edge of 
the cover-glass and drawn underneath it by the suction of 
a piece of blotting-paper touched to the opposite edge; 
or a small drop of the reagent and of the urine may be 
placed close together upon a slide and a cover gently 
lowered over them. As the two fluids mingle, the effect 
upon various structures may be seen. 



MICROSCOPIC EXAMINATION 



141 



Urinary sediments may be studied under three heads: 
A. Unorganized sediments. B. Organized sediments. C. 
Extraneous structures. 



A. Unorganized Sediments 
In general these have little diagnostic or prognostic 
significance. Most of them are substances normally 
present in solution, which have been precipitated either 
because present in excessive amounts, or, more frequently, 
because of some alteration in the urine (as in reaction, 
concentration, etc.) which may be purely physiologic, 
depending upon changes in diet or habits. Various 
substances are always precipitated during decompo- 
sition, which may take place either within or without 




Fig. 36. — Unusual urinary crystals (drawn from various authors): i, Calcium sul- 
phate (colorless); 2, cholesterin (colorless); 3, hippxiric acid (colorless); 4, hematoidin 
(brown); 5, fatty acids (colorless); 6, indigo (blue); 7, sodium urate (yellowish). 

the body. Unorganized sediments may be classified 
according to the reaction of the urine in which they are 
most likely to be found : 

In acid urine: Uric acid, amorphous urates, sodium 
urate, calcium oxalate, leucin and ty rosin, cystin, and 
fat-globules. Uric acid, the urates, and calcium oxalate 



142 



THE URINE 



are the common deposits of acid urines; the others are 
less frequent, and depend less upon the reaction of the 
urine. 

In alkaline urine: Phosphates, calcium carbonate, and 
ammonium urate. 

Other crystalline sediments (Fig. 36) which are rare 
and require no further mention are: Calcium sulphate, 
cholesterin, hippuric acid, hematoidin, fatty acids, and 
indigo. 




Fig- 37- — Forms of uric acid: i, Rhombic plates; 2, whetstone forms; 3, 3, quadrate 
forms; 4, 5. prolonged into points; 6, 8, rosets; 7. pointed bundles; g, barrel forms pre- 
cipitated by adding hydrochloric acid to urine (Ogden). 

I. In Acid Urine.— (i) Uric-acid Crystals.— These 

crystals are the red grains — " gravel " or " red sand " — 
which are often seen adhering to the sides and bottom 
of a vessel containing urine. ]VIicroscopIcally, they are 
yellow or reddish-brown crystals, which differ greatly in 



PLATE III 




Uric-acid crystals with amorphous urates (after Payer). 



MICROSCOPIC EXAMINATION 143 

size and shape. The most characteristic forms (Plate III 
and Fig. 37) are "whetstones"; roset-like clusters of 
prisms and whetstones; and rhombic plates, which 
have usually a paler color than the other forms and are 
sometimes colorless. A very rare form is a colorless 
hexagonal plate resembling cystin. Recognition of the 
crystals depends less upon their shape than upon their 
color, the reaction of the urine, and the facts that they 
are soluble in caustic soda solution and insoluble in hy- 
drochloric or acetic acid. When ammonia is added, 
they dissolve and crystals of ammonium urate appear. 

A deposit of uric-acid crystals has no significance un- 
less it occurs before or very soon after the urine is voided. 
Every urine, if kept acid, will in time deposit its uric 
acid. Factors which favor an early deposit are high 
acidity, diminished urinary pigments, and excessive ex- 
cretion of uric acid. The chief clinical interest of the 
crystals lies in their tendency to form calculi, owing to 
the readiness with which they collect about any solid 
object. Their presence in the freshly voided urine in 
clusters of crystals suggests stone in the kidney or 
bladder, especially if blood is also present. (See Fig. 65.) 

(2) Amorphous Urates. — These are chiefly urates of 
sodium and potassium which are thrown out of solution 
as a yellow or red " brick-dust " deposit. In pale 
urines this sediment is almost white. It disappears upon 
heating. A deposit of amorphous urates is very common 
in concentrated and strongly acid urines, especially in 
cold weather, and has no clinical significance. Under 
the microscope it appears as fine yellowish granules, 
often so abundant as to obscure all other structures 
(Plate III). In such cases the urine should be warmed 



144 THE URINE 

before examining. Amorphous urates are readily sol- 
uble in caustic soda solutions. When treated with hy- 
drochloric or acetic acid, they slowly dissolve and rhombic 
crystals of uric acid appear. 

Rarely, sodium urate occurs in crystalline form — 
slender prisms, arranged in fan- or sheaf-hke structures 
(Fig. 36). 

(3) Calcium Oxalate. — Characteristic of calcium oxa- 
late are colorless, glistening, octahedral crystals, giving 
the appearance of small squares crossed by two intersect- 



ffi 



^ 




Fig. 38. — Various forms of calcium oxalate crystals (Ogden). 

ing diagonal lines — the so-called " envelop crystals " 
(Fig. 51). They vary greatly in size, being sometimes 
so small as to seem mere points of light with medium- 
power objectives. Unusual forms, which, however, 
seldom occur except in conjunction with the octahedra, 
are colorless dumb-bells, spheres, and variations of the 
octahedra (Fig. 38). The spheres might be mistaken for 
globules of fat or red blood-corpuscles. Crystals of 
calcium oxalate are insoluble in acetic acid or caustic 
soda. They are dissolved by strong hydrochloric acid, 



MICROSCOPIC EXAMINATION I45 

and recrystallize as octahedra upon addition of ammonia. 
They are sometimes encountered in alkaline urine. 

The crystals are commonly found in the urine after 
ingestion of vegetables rich in oxaHc acid, as tomatoes, 
spinach, asparagus, and rhubarb. They have no de- 
finite significance pathologically. They often appear 
in digestive disturbances, in neurasthenia, and when the 
oxidizing power of the system is diminished. When 
abundant, they are generally associated with a little 
mucus; and, in men, frequently with a few spermatozoa. 
Like uric acid, their chief clinical interest lies in their 
tendency to form calculi, and their presence in fresh 
urine, together with evidences of renal or cystic irritation, 
should be viewed with suspicion, particularly if they are 
clumped in small masses. 

(4) Leucin and Tyrosin. — Crystals are deposited only 
when the substances are present in considerable amount. 
When present in smaller amount, they will usually be 
deposited if a little of the urine be slowly evaporated upon 
a slide. Addition of alcohol favors the deposit. They 
generally appear together, and are of comparatively 
rare occurrence, usually indicating severe fatty destruc- 
tion of the liver, such as occurs in acute yellow atrophy 
and phosphorus-poisoning. 

The crystals cannot be identified from their morphol- 
ogy alone, since other substances, notably calcium phos- 
phate (Fig. 42) and ammonium urate, may take similar 
or identical forms. 

Leucin crystals (Fig. 39) as they appear in the urine 

do not represent the pure substance. They are slightly 

yellow, oily-looking spheres, many of them with radial 

and concentric striations. Some may be merged to- 

10 



146 THE URINE 

gether in clusters. They are not soluble in hydrochloric 
acid nor in ether. 

Tyrosin crystallizes in very fine colorless needles, 
usually arranged in sheaves, with a marked constriction 
at the middle (Fig. 39). It is soluble in ammonia and 
hydrochloric acid, but not in acetic acid. 

(5) Cystin crystals are colorless, highly refractive, 
rather thick, hexagonal plates with well-defined edges. 
They lie either singly or superimposed to form more or 
less irregular clusters (Fig. 40). Uric acid sometimes 




Fig. 3Q. — Leucin spheres and tyrosin needles (Stengel). 

takes this form and must be excluded. Cystin is soluble 
in hydrochloric acid, insoluble in acetic; it is readily 
soluble in ammonia and recrystallizes upon addition of 
acetic acid. 

Cystin is one of the amino-acids formed in decompo- 
sition of the protein molecule, and is present in traces in 
normal urine. Crystals are deposited only when the sub- 
stance is present in excessive amount. Their presence 
is known as cystiniiria. It is a rare condition due to an 
obscure abnormality of protein metabolism and usually 



MICROSCOPIC EXAMINATION 



147 



continues throughout life. There are rarely any symp- 
toms save those referable to renal or cystic calculus, to 
which the condition strongly predisposes. 

(6) Fat-globules. — Fat appears in the urine as highly 
refractive globules of various sizes, frequently very small. 
These globules are easily recognized from the fact that 
they are stained black by osmic acid and orange or red 
by Sudan III. The stain may be applied upon the slide, 




Fig. 40. — Cystin crystals from urine of patient with cystin calculus ( X 200) (photograph bj 

the author). 



as already described (p. 140). Osmic acid should be 
used in i per cent, aqueous solution ; Sudan III in satu- 
rated solution in 70 per cent, alcohol, to which one-half 
volume of 10 per cent, formalin may advantageously 
be added. 

Fat in the urine is usually a contamination from un- 
clean vessels, oiled catheters, etc. A very small amount 
may be present after ingestion of large quantities of cod- 



148 THE URINE 

liver oil or other fats. In fatty degeneration of the 
kidney, as in phosphorus-poisoning and chronic paren- 
chymatous nephritis, fat-globules are commonly seen, 
both free in the urine and embedded in cells and tube- 
casts. 

In chyluria, or admixture of chyle with the urine as a 
result of rupture of a lymph-vessel, minute droplets of 
fat are so numerous as to give the urine a milky appear- 
ance. The droplets are generally smaller than those of 
milk. The fluid is often blood-tinged. Chyluria occurs 
most frequently as a symptom of infection by filaria 
(p. 357), the embryos of which can usually be found in 
the milky urine. 

2. In Alkaline Urine.— (i) Phosphates.— While most 
common in alkaline urine, phosphates are sometimes 
deposited in amphoteric or feebly acid urines. The usual 
forms are: {a) Ammoniomagnesium phosphate crystals; 
{h) acid calcium phosphate crystals; and (c) amorphous 
phosphates. All are readily soluble in acetic acid. 

{a) Ammonioynagnesium Phosphate Crystals. — They 
are the common " triple phosphate " crystals, which are 
generally easily recognized (Figs. 41 and 66. and Plate 
IV). They are colorless, except when bile-stained. 
Their usual form is some modification of the prism, with 
oblique ends. Alost tx-pical are the well-known '* coffin- 
Hd " and '' hip-roof " forms. The long axis of the hip- 
roof crystal is often so shortened that it resembles the 
envelop crystal of calcium oxalate. It does not, how- 
ever, have the same luster; this, and its solubility in acetic 
acid, will always prevent confusion. 

When rapidly deposited, as by artificial precipitation, 
triple phosphate often takes feathery, star-, or leaf-like 



MICROSCOPIC EXAMINATION 



149 



forms. These gradually develop into the more common 
prisms. X-forms may be produced by partial solution of 
prisms. 

(b) Acid Calcium Phosphate Crystals.- — In feebly acid, 
amphoteric, or feebly alkaline urines acid calcium phos- 
phate, wrongly called " neutral calcium phosphate," 
is not infrequently deposited in the form of colorless 
prisms arranged in stars and rosets (Fig. 42, i). The 
individual prisms are usually slender, with one beveled, 




Fig. 41. — Various forms of triple phosphate crystals (Ogden). 

wedge-like end, but are sometimes needle-like. They 
may sometimes take forms resembling tyrosin (Fig. 42, 
2), calcium sulphate, or hippuric acid, but are readily 
distinguished by their solubility in acetic acid. 

Calcium phosphate often forms large, thin, irregular, 
usually granular, colorless plates, which are easily recog- 
•nized (Fig. 42, 3). 

(c) Amorphous Phosphates. — The earthy phosphates 
are thrown out of solution in most alkaline and many 
amphoteric urines as a white, amorphous sediment, 



150 



THE URINE 



which may be mistaken for pus macroscopically. Under 
the microscope the sediment is seen to consist of numer- 
ous colorless granules, distinguished from amorphous 




V 




Fig. 42. — Crystals of calcium phosphate: i, Common form Ccopied from Rieder's 
Atlas); 2, needles resembling tyrosin (drawn from nature); 3, large, irregular plates ({rem 
nature). 

urates by their color, their solubility in acetic acid, and 
the reaction of the urine. 

The various phosphatic deposits frequently occur 
together. They are sometimes due to excessive excre- 






9= ' ff'^%^ 






•^ .^^^ 



^'^^ 



*d? 



Fig. 43. — Indistinct crystalline sediment (dumb-bell crystals) of calcium carbonate. 
Similar crystals are formed by calcium oxalate and calcium sulphate (after Funke). 

tion of phosphoric acid, but usually merely indicate that 
the urine has become, or is becoming, alkaline. (See 
Phosphates, p. 86.) 

(2) Calcium carbonate may sometimes be mingled 
with the phosphatic deposits, usually as amorphous 



PLATE IV 




Sediment of alkaline fermentation (after Hofmann and Ultzmann). 



MICROSCOPIC EXAMINATION 151 

granules, or, more rarely, as -cfelorl^sg^Sjph^res ^nd dumb- 
bells (Fig. 43), which are soXu\:)^(^kh 9x^)Ac acici wi'tli ^^s/*. Tii 
formation. ' ' ' ^ ' ^ ' ' ff« 

(3) Ammonium Urate Crystals. — This is the only 
urate deposited in alkaline urine. It forms opaque 
yellow crystals, usually in the form of spheres (Plate IV. 
and Fig. 66) , which are often covered with fine or coarse 




Fig. 44.— Crystals of ammonium urate (one-half of the forms copied from Rieder's Atlas, 
the others from nature). 

spicules — " thorn-apple crystals." Sometimes dumb- 
bells, compact sheaves of fine needles, and irregular 
rhizome forms are seen (Fig. 44). Upon addition of 
acetic acid they dissolve, and rhombic plates of uric acid 
appear. 

These crystals occur only when free ammonia is 
present. They are generally found along with the phos- 
phates in decomposing urine and have no clinical 
significance. 

B. Organized Sediments 
The principal organized structures in urinary sedi- 
ments are: Tube-casts; epithelial cells; pus-corpuscles; 



152 THE URINE 

red bIood-corpusde>^ ; sj^ennatozoa ; bacteria, and animal 
parasites. They are njuck more important than the 
unorganized sediments just considered. 

1 . Tube=casts. — These interesting structures are albu- 
minous casts of the uriniferous tubules. Their pres- 
ence in the urine probably always indicates some 
pathologic change in the kidney, although this change 
may be very slight or transitory. Large numbers may 
be present in temporary irritations and congestions. 
They do not in themselves, therefore, imply organic dis- 
ease of the kidney. They rarely occur in urine which 
does not contain, or has not recently contained, al- 
bumin. 

While it is not possible to draw a sharp dividing-line 
between the different varieties, casts may be classified 
as follows: 

(i) Hyaline casts. 

(a) Narrow. 

(b) Broad. 

(2) Waxy casts. 

(3) Fibrinous casts. 

(4) Granular casts. 

(a) Finely granular. 
(h) Coarsely granular. 

(5) Fatty casts. 

(6) Casts containing organized structures. 

((/) Epithelial casts. 

(b) Blood-casts. 

(c) Pus-casts. 

(d) Bacterial casts. 

As will be seen later, practically all varieties are 
modifications of the hyaline. 



MICROSCOPIC EXAMINATION 1 53 

The significance of the different varieties is more 
readily understood if one considers their mode of forma- 
tion. Albuminous material, the source, and nature of 
which are not definitely known, but which are doubtless 
not the same in all cases, probably enters the lumen of a 
uriniferous tubule in a fluid or plastic state. The 
material has been variously thought to be an exudate 
from the blood, a pathologic secretion of the renal cells, 
and a product of epithelial degeneration. In the tubule 
it hardens into a cast which, when washed out by the 
urine, retains the shape of the tubule, and contains within 
its substance whatever structures and debris were lying 
free within the tubule or were loosley attached to its wall. 
If the tubule be small and have its usual lining of epithe- 
lium, the cast will be narrow; if it be large or entirely 
denuded of epithelium, the cast will be broad. A cast, 
therefore, indicates the condition oj the tubule in which 
it is formed, but does not necessarily indicate the condition 
of the kidney as a whole. 

The search for casts must be carefully made. The 
urine must be fresh, since hyaline casts soon dissolve 
when it becomes alkaline. It should be thoroughly 
centrifugalized. When the sediment is abundant, casts, 
being light structures, will be found near the top. In 
cystitis, where casts may be entirely hidden by the pus, 
the bladder should be irrigated to remove as much of 
the pus as possible and the next urine examined. In 
order to prevent solution of the casts the urine, if al- 
kaline, must be rendered acid by previous administra- 
tion of boric acid or other drugs. Heavy sediments of 
urates, blood, or vaginal cells may likewise obscure 
casts and other important structures. The last can be 



154 



THE URINE 



avoided by catheterization. Urates can be dissolved 
by gently warming before centrifugalizing, care being 
taken not to heat enough to coagulate the albumin. 
The albumin shield of .the centrifuge tube may also 
be heated. Blood can be destroyed by centrifugalizing, 
pouring off the supernatant urine, tilling the tube with 
water, adding a few drops of dilute acetic acid, mixing 
well, and again centrifugalizing; this process being 
repeated until the blood is completely decolorized. 
Too much acetic acid will dissolve hyaline casts. 

Their cylindric shape can be best seen by slightly 
moving the cover-glass w^hile observing them, thus 
causing them to roll. This little manipulation should 
be practised until it can be done satisfactorily. It will 
prove useful in many examinations. 

Various methods of staining casts so as to render them 
more conspicuous have been proposed. They offer no 
special advantage to one who understands how to use 
the substage mechanism of his microscope. The " nega- 
tive-staining ■' method is as good as any. It consists 
simply in adding a little India-ink to the drop of urine on 
the slide. Casts, cells, etc., will stand out as colorless 
structures on a dark background. 

(i) Hyaline Casts. — Typically, these are colorless, 
homogeneous, semitransparent, cyhndric structures, with 
parallel sides and usually rounded ends. Not infre- 
quently they are more opaque or show a few granules or 
an occasional oil-globule or cell, either adhering to them 
or contained within their substance. Generally they 
are straight or curved; less commonly, convoluted. 
Their length and breadth vary greatly : they are some- 
times so long as to extend across several fields of a 



MICROSCOPIC EXAMINATION 



155 



medium-power objective, but are usually much shorter; 
in breadth, they vary from one to seven or eight times 




Fig. 45. — Hyaline casts showing fat-droplets and leukocytes (obj. one-sixth) (Boston). 



the diameter of a red blood-corpuscle. (See Figs. 4, 45, 
46, and 50.) 




Fig. 46. — Various kinds of casts: a. Hyaline and finely granular cast; b, finely granular 
cast; c, coarsely granular cast; d, brown granular cast; e, granular cast with normal and 
abnormal blood adherent; /, granular cast with renal cells adherent; ;;, granular cast with 
fat and a fatty renal cell adherent COgden). 

Hyaline casts are the least significant of all the casts, 
and occur in many slight and transitory conditions. 



156 THE URINE 

Small numbers are common following ether anesthesia, 
in fevers, after excessive exercise, and in congestions and 
irritations of the kidney. They are always present, and 
are usually stained yellow when the urine contains much 
bile. While they are found in all organic diseases of the 
kidney, they are most important in chronic interstitial 
nephritis. Here they are seldom abundant, but their 
constant presence is the most reliable urinary sign of the 
disease. Small areas of chronic interstitial change are 
probably responsible for the few hyaline casts so fre- 
quently found in the urine of elderly persons. 




Fig. 47. — Waxy casts (upper part of figure). Fatty and fat-bearing casts (lower part of 
figure) (from Greene's "Medical Diagnosis"). 

Very broad hyaline casts commonly indicate complete 
desquamation of the tubular epithelium, such as occurs 
in the late stages of nephritis. 

(2) Waxy Casts. — Like hyaline casts, these are homo- 
geneous when typical, but frequently contain a few 
granules or an occasional cell. They are much more 
opaque than the hyaline variety, and are usually shorter 
and broader, with irregular, broken ends, and some- 
times appear to be segmented. They are grayish or 
colorless, and have a dull, waxy look, as if cut from par- 



MICROSCOPIC EXAMINATION 



157 



aflSn (Figs. 47 and 64). They are sometimes composed 
of material which gives the amyloid reactions. Waxy 
casts are found in most advanced cases of nephritis, 
where they are an unfavorable sign. They are perhaps 
most frequently found in amyloid disease of the kidney, 
but are not distinctive of the disease, as is sometimes 
stated. 

(3) Fibrinous Casts. — Casts which resemble waxy 
casts, but have a distinctly yellow color, as if cut from 
beeswax, are often seen in acute nephritis. They are 




Fig. 48. — Granular and fatty casts and two compound granular cells (Stengel). 

called fibrinous casts, but the name is inappropriate, as 
they are not composed of fibrin. They are often classed 
with waxy casts, but should be distinguished, as their 
significance is much less serious. 

(4) Granular Casts. — These are merely hyaline casts 
in which numerous granules are embedded (Figs. 46, 48, 
and 50). 

Finely granular casts contain many fine granules, are 
usually shorter, broader, and more opaque than the 
hyaline variety, and are more conspicuous. Their color 
is grayish or pale yellow. 



158 THE URINE 

Coarsely granular casts contain larger granules and are 
darker in color than the finely granular, being often dark 
brown owing to presence of altered blood-pigment. They 
are usually shorter and more irregular in outline, and 
more frequently have irregularly broken ends. 

(5) Fatty Casts. — Small droplets of fat may at times 
be seen in any variety of cast. Those in which the drop- 
lets are numerous are called fatty casts (Figs. 47 and 48) . 
The fat-globules are not difficult to recognize. Staining 
with osmic acid or Sudan (p. 147) will remove any doubt 
as to their nature. 

The granules and fat-droplets seen in casts are prod- 
ucts of epithelial degeneration. Granular and fatty 
casts, therefore, always indicate partial or complete dis- 
integration of the renal epithelium. The finely granular 
variety is the least significant, and is found when 
the epithelium is only moderately affected. Coarsely 
granular, and especially fatty casts, if present in con- 
siderable numbers, indicate a serious parenchymatous 
nephritis. 

(6) Casts Containing Organized Structures. — Cells 
and other structures are frequently seen adherent to a 
cast or embedded within it. (See Figs. 45 and 46). 
When numerous, they give name to the cast. 

(a) Epithelial casts contain epithelial cells from the 
renal tubules. They always imply desquamation of 
epithelium, which rarely occurs except in parenchy- 
matous inflammations (Figs. 63 and 64). When the 
cells are well preserved they point to acute nephritis. 

ib) Blood-casts contain red blood-corpuscles, usually 
much degenerated (Figs. 49 and 63). They always 
indicate hemorrhage into the tubules, which is most 



MICROSCOPIC EXAMINATION 



159 



common in acute nephritis or an acute exacerbation of 
a chronic nephritis. 

(c) Pus-casts (see Fig. 65), composed almost wholly of 
pus-corpuscles, are uncommon, and point to a chronic 
suppurative process in the kidney. 

(d) True bacterial casts are rare. They indicate a 
septic condition in the kidney. Bacteria may permeate 
a cast after the urine is voided. 




Fig. 49. — Red blood-corpuscles and blood-casts (courtesy of Dr. A. Scott) (obj. one- 
sixth) (Boston) 

Structures Likely to be Mistaken for Casts. — (i) 
Mucous Threads. — Mucus frequently appears in the 
form of long strands which slightly resemble hyaline 
casts (Fig. 50). They are, however, more ribbon-like, 
have less well-defined edges, and usually show faint 
longitudinal striations. Their ends taper to a point or 
are split or curled upon themselves, and are never evenly 
rounded, as is commonly the case with hyaline casts. 

Such threads form a part of the nubecula of normal 
urine, and are especially abundant when calcium oxalate 



i6o 



THE URINE 



crystals are present. When there is an excess of mucus, 
as in irritations of the urinary tract, every field may be 
filled with an interlacing meshwork. 

Mucous threads are microscopic and should not be 
confused with urethral shreds, which are macroscopic, 
and consist of a matrix of mucus in which many epi- 
thelial and pus-cells are embedded. 

(2) Cylindroids. — This name is sometimes given to the 
mucous threads just described, but is more properly 




Fig. so. — Hyaline and granular casts, mucous threads, and cylindroids. There are also 
a few epithelial cells from the bladder (Wood). 

applied to certain peculiar structures more nearly allied 
to casts. They resemble hyaline casts in structure, but 
differ in being broader at one end and tapering to a 
slender tail, which is often twisted or curled upon itself 
(Fig. 50). They frequently occur in the urine along 
with hyaline casts, especially in irritations of the kidney, 
and have no definite pathologic significance. 

(3) Masses of amorphous urates, or phosphates, or 



MICROSCOPIC EXAMINATION l6l 

very small crystals (Fig. 51), which accidentally take a 
cylindric form, or shreds of mucus covered with granules, 
closely resemble granular casts. Application of gentle 
heat or appropriate chemicals will serve to differentiate 
them. When urine contains both mucus and granules, 
large numbers of these " pseudocasts," all lying in the 
same direction, can be produced by slightly moving the 
cover-glass from side to side. It is possible — as in urate 
infarcts of infants — for urates to be molded into cylin- 
dric bodies within the renal tubules. 




Fig. SI. — Calcium oxalate crystals, showing a pseudocast of small crystals (Jakob). 

(4) Hairs and fibers of wool, cotton, etc. These 
could be mistaken for casts only by beginners. One 
can easily become familiar with their appearance by 
suspending them in water and examining with the micro- 
scope (Fig. 61). 

(5) Hyphae of molds are not infrequently mistaken 
for hyaline casts. Their higher degree of refraction, 
their jointed or branching structure, and the accom- 
panying spores will differentiate them (Fig. 62). 

11 



1 62 



THE URINE 



2. Epithelial Cells.— A few cells from various parts 
of the urinary tract occur in every urine. A marked 
increase indicates some pathologic condition at the site of 
their origin. It is sometimes, but by no means always, 
possible to locate their source from their form. Most 
cells are much altered from their original shape. Any 
epithelial cell may be so granular from degenerative 
changes that the nucleus is obscured. They are usually 
divided into three groups: 

(i) Small, round or polyhedral 
^mW^£f cells are about the size of pus- 

^k^m corpuscles, or a little larger, with a 

v^tfft «... single round nucleus. Such cells 

may come from the deeper layers 
of any part of the urinary tract. 
They are uncommon in normal 
urine. When they are dark in 
color, very granular, and contain 
a comparatively large nucleus, they 
probably come from the renal tub- 
ules, but their origin in the kid- 
ney is not proved unless they are 
found embedded in casts. Renal 
cells are abundant in parenchyma- 
tous nephritis, especially the acute form. They are 
nearly always fatty — most markedly so in chronic paren- 
chymatous nephritis, where their substance is sometimes 
wholly replaced by fat-droplets (" compound granule 
cells ") (see Figs. 48, 52, and 63). 

(2) Irregular cells are considerably larger than the 
preceding. They are round, pear shaped, or spindle 
shaped, or may have tail-like processes, and are hence 




Fig. S2- — Renal epithelium 
from nephritic urine: a, Poly- 
hedral epithelium in nephritis 
of scarlet fever; b and c, differ- 
ent grades of fatty degenera- 
tion in renal epithelium in 
chronic nephritis ( X 400) (after 
Bizzozero). 



MICROSCOPIC EXAMINATION 



163 



named large round, pyriform, spindle, or caudate cells 
respectively. Each contains a round or oval distinct 
nucleus. Their usual source is the deeper layers of the 
urinary tract, especially of the bladder. Caudate forms 
come most commonly from the pelvis of the kidney (see 
Figs. 53, h, 54, 65, and 66). 

(3) Squamous or pavement cells are large flat cells, 
each with a small, distinct, round or oval nucleus (Fig. 
53, a). They are derived from the superficial layers of 




Fig- 53- — Epithelial cells from urethra and bladder: a. Squamous cells from superficia' 
layers; b, irregular cells from deeper layers (Jakob). 



the ureters, bladder, urethra, or vagina, and when 
desquamation is active, appear in stratified masses. 
Squamous cells from the bladder are generally rounded, 
while those from the vagina are larger, thinner, and 
more angular. Great numbers of these vaginal cells, 
"together with pus-corpuscles, may be present when 
leukorrhea exists. 

3. Pus=corpuscles.— A very few leukocytes are pres- 
ent in normal urine. They are more abundant when 



i64 



THE URINE 



mucus is present. An excess of leukocytes, mainly of 
the polymorphonuclear variety, with albumin, consti- 
tutes pyuria — pus in the urine. 




Fig. 54. — Caudate epithelial cells from pelvis of kidney (Jakob). 

When at all abundant, pus forms a w^iite sediment 
resembling amorphous phosphates macroscopically. Un- 



® ® ® 
® 



® ® ® 



Fig- 55- — Pus<orpuscles: a, .\s ordinarily seen; h, ameboid corpuscles; c, showing the 
action of acetic acid (Ogden). 



der the microscope the corpuscles appear as very granu- 
lar cells, about twice the diameter of a red blood-cor- 
puscle (Figs. 55 and 66). In freshly voided urine many 



MICROSCOPIC EXAMINATION 1 65 

exhibit ameboid motion, assuming irregular outlines. 
Each contains one irregular nucleus or several small, 
rounded nuclei. The nuclei are obscured or entirely 
hidden by the granules, but may be brought clearly 
into view by running a httle acetic acid under the cover- 
glass. This enables one to easily distinguish pus-cor- 
puscles from small round epithelial cells, which resemble 
them in size, but have a single, rather large, round 
nucleus. In decomposing urine pus is converted into a 
gelatinous mass which gives the urine a ropy consistence. 

Pyuria indicates suppuration in some part of the 
urinary tract — urethritis, cystitis, pyelitis, etc. — or may 
be due to contamination from the vagina, in which case 
many vaginal epithelial cells will also be present. In 
general, the source of the pus can be determined only by 
the accompanying structures (epithelia, casts) or by the 
clinical signs. 

A fairly accurate idea of the quantity of pus from day 
to day may be had by shaking the urine thoroughly and 
counting the number of corpuscles per cubic millimeter 
upon the Thoma-Zeiss blood-counting slide. 

4. Red Blood=corpuscles.— Urine which containsblood 
is always albuminous. Very small amounts do not alter 
its macroscopic appearance. Larger amounts alter it 
considerably. Blood from the kidneys is generally 
intimately mixed with the urine and gives it a hazy 
reddish or brown color. When from the lower urinary 
tract, it is not so intimately mixed and settles more 
quickly to the bottom, the color is brighter, and small 
clots are often present. 

Red blood-corpuscles are not usually difficult to recog- 
nize with the microscope. When very fresh, they have a 



1 66 THE URINE 

normal appearance, being yellowish discs of uniform size 
(normal blood). When they have been in the urine any 
considerable time, their hemoglobin may be dissolved out, 
and they then appear as faint colorless circles or " shadow 
cells " (abnormal blood), and are more difficult to see 
(Fig. 56; see also Figs. 49 and 63). They are apt to be 
swollen in dilute and crenated in concentrated urines. 
The microscopic findings may be corroborated by chemic 
tests for hemoglobin, although the microscope may show 
a few red corpuscles when the chemic tests are negative. 
When not due to contamination from menstrual dis- 
charge, blood in the urine, or hematuria, is always patho- 






O ^ O o o - 



Fig. 56. — Blood-corpuscles: a, Normal; h, abnormal (Ogden). 

logic. Blood comes from the kidney tubules in severe 
h^^peremia, in acute nephritis and acute exacerbations of 
chronic nephritis, and in renal tuberculosis and malig- 
nant disease. An " idiopathic hematuria," probably of 
nervous origin, has been observed. The finding of blood- 
casts is the only certain means of diagnosing the kidney 
as its source. Blood comes from the pelvis of the kidney 
in renal calculus (Fig. 65), and is then usually intermit- 
tent, small in amount, and accompanied by a Httle pus 
and perhaps crystals of the substance forming the stone. 
Considerable hemorrhages from the bladder may occur 
in vesical calculus, tuberculosis, and new growths. 
Small amounts of blood generally accompany acute 



MICROSCOPIC EXAMINATION 



167 



cystitis. In Africa the presence of Schistosomum hema- 
tobium in the veins of the bladder is a common cause of 
hemorrhage (Egyptian hematuria) . 

5. Spermatozoa are generally present in the urine of 
men after nocturnal emissions, after epileptic convul- 
sions, and in spermatorrhea. They may be found in the 
urine of both sexes following coitus. They are easily 
recognized from their characteristic structure (Fig. 57). 



r "^ 


y 









© 






^ 


r. 





(, 


J 
i 


?i 


C 








c 



9 




p 


1 
^^ 























Fig. S7- — Sjjermatozoa in urine (Ogden). 



The 4 mm. objective should be used, with subdued light 
and careful focusing. 

6. Bacteria. — Normal urine is free from bacteria in 
the bladder, but becomes contaminated in passing 
through the urethra. Various non-pathogenic bacteria, 
notably Micrococcus urece (Fig. 58), are always present 
in decomposing urine. In suppurations of the urinary 
tract pus-producing organisms may be found. In many 



1 68 THE URINE 

infectious diseases the specific bacteria may be eliminated 
in the urine without producing any local lesion. Ty- 
phoid bacilli have been known to persist for months 
and even years after the attack. 

Bacteria produce a cloudiness which will not clear 
upon filtration. They are easily seen with the 4 mm. 
objective in the routine microscopic examination. 
Ordinarily, no attempt is made to identify any but the 
tubercle bacillus and the gonococcus. 



Fig. 58. — Micrococcus urese (after von Jaksch). 

Tubercle bacilli are nearly always present in the urine 
when tuberculosis exists in any part of the urinary tract, 
but are often difficult to find, especially when the urine 
contains little or no pus. 



Detection of Tubercle Bacilli in Urine. — The urine should 
be obtained by catheter after careful cleansing of the parts. 

(i) Centrifugalize thoroughly, after dissolving any sediment 
of urates or phosphates by gentle heat or acetic add. Pour 
off the supernatant fluid, add water, and centrifugalize again. 
Addition of one or two volumes of alcohol will favor cen- 
trifugalization by lowering the specific gravity. 

(2) Make thin smears of the sediment, adding a little egg- 
albumen if necessary to make the smear adhere to the glass; 
dry. and fix in the usual way. 

(3) Stain with carbol-fuchsin, steaming for at least three 
minutes, or at room temperature for six to twelve Hours. 



■^ 



PLATE V 



% 
% 






/) It 




Tubercle bacilli in urinary sediment; X 800 (Ogden). 



MICROSCOPIC EXAMINATION 1 69 

(4) Wash in water, and then in 20 per cent, nitric acid 
until only a faint pink color remains. 

(5) Wash in water. 

(6) Soak in alcohol fifteen minutes or longer. This decolor- 
izes the smegma bacillus (p. 53), which is often present in 
the urinCj^ and might easily be mistaken for the tubercle bacil- 
lus. It is unlikely, however, to be present in catheterized 
specimens. It is always safest to soak the smear in alcohol 
for several hours or over night, since some strains of the smeg- 
ma bacillus are very resistant. 

(7) Wash in water. 

(8) Apply Loffler's methylene-blue solution one-half minute. 

(9) Rinse in water, dry between filter-papers, and examine 
with the one-twelfth objective. 

When the bacilli are scarce, the following method may be 
tried. It is applicable also to other fluids. If the fluid is not 
albuminous, add a little egg-albumen. Coagulate the albu- 
men by gentle heat and centrifugalize. The bacilli will be 
carried down with the albumen. The sediment is then treated 
by the antiformin method (p. 52). 

A careful search of many smears may be necessary to find 
the bacilli. They usually lie in clusters (see Plate V). Fail- 
ure to find them in suspicious cases should be followed by 
inoculation of guinea-pigs; this is the court of last appeal, 
and must also be sometimes resorted to in order to exclude 
the smegma bacillus. 

In gonorrhea gonococci are sometimes found in 
the sediment, but more commonly in the " gonorrheal 
threads," or " floaters." In themselves, these threads 
are by no means diagnostic of gonorrhea. Detection of 
the gonococcus is described later (p. 369). 

7. Animal parasites are rare in the urine. Booklets 
and scolices of Tcenia echinococcus (Fig. 59) and em- 



170 



THE URINE 



bryos pf filariae have been met. In Africa the ova, and 
even adults, of Schistosomum hcemalobium are common, 




Fig. 5g. — I, Scolcx of ta-nia cchinococcus. showin;; crown of booklets; 2, scolex and 
detached booklets (obj. one-sixth) (Boston). 



accompanying " Egyptian hematuria." Trichomonas 
vaginalis is a not uncommon contamination. This and 




Fig. 60. — Embryo of "vinegar eel" in urine, from contamination; length. 340 Mi 
width, IS ix. .\n epithelial cell from bladder and three leukocytes are also shown (studied 
with Dr. J. .\ Wilder). 

other protozoa may be mistaken for spermatozoa by the 
inexperienced. 



MICROSCOPIC EXAMINATION 171 

A worm which is especially interesting is Anguillula 
aceti, the "vinegar eel." This is generally present in 
the sediment of table vinegar, and may reach the urine 
through use of vinegar in vaginal douches, or through 
contamination of the bottle in which the urine is con- 
tained. It has been mistaken for Sirongyloides intes- 
tinalis and for the filaria embryo. It closley resembles 
the former in both adult and embryo stages. The young 
embryos have about the same length as filaria embryos, 
but are nearly twice as broad and the intestinal canal is 
easily seen (compare Figs. 60 and 134). For fuller de- 
scriptions of these parasites the reader is referred to 
Chapter VI. 

C. Extraneous Structures 

The laboratory worker must familiarize himself with 
the microscopic appearance of the more common of the 
numerous structures which may be present from acci- 
dental contamination (Fig. 61). 

Yeast-cells are smooth, colorless, highly refractive, 
spheric or ovoid cells. They sometimes reach the size of 
a leukocyte, but are generally smaller (see Fig. 106, I). 
They might be mistaken by the inexperienced for red 
blood-corpuscles, fat-droplets, or the spheric crystals of 
calcium oxalate, but are distinguished by the facts that 
they are not of uniform size; that they tend to adhere in 
short chains; that small buds may often be seen ad- 
hering to the larger cells; and that they do not give the 
hemoglobin test, are not stained by osmic acid or Sudan, 
but are colored brown by Lugol's solution, and are in- 
soluble in acids and alkalis. Yeast-cells multiply rapidly 



172 



THE URINE 



in diabetic urine, and may reach the bladder and multi- 
ply there. 

Mold fungi (Fig. 62) are characterized by refractive, 
jointed, or branched rods (hyphae), often arranged in a 
network, and by highly refractive, spheric or ovoid spores. 




Fig. 61. — Extraneous matters found in urine: a. Flax-fibers; b, cotton-fibers; c. feathers; 
d, hairs; e, potato-starch; /, rice-starch granules; g, wheat-starch; h, air-bubbles; », 
muscular tissue; k, vegetable tissue; /, oil-globules. 



They are common in urine which has stood exposed to 
the air. 

Fibers of wool, cotton, linen, or silk, derived from 
towels, the clothing of the patient, or the dust in the air, 
are present in almost every urine. Fat-droplets are most 
frequently derived from unclean bottles or oiled cathe- 



THE URINE IN DISEASE 1 73 

ters. Starch-granules may reach the urine from towels, 
the clothing, or dusting-powders. They are recognized 
by their concentric striations and their blue color with 
iodin solution. Lycopodium granules (Fig. 5) may also 
reach the urine from dusting-powders. They might be 
mistaken for the ova of parasites. Bubbles of air are 
often confusing to beginners, but are easily recognized 




Fig. 62. — Aspergill»is from urine (Boston). 

after once being seen. Scratches and flaws in the glass 
of slide or cover are likewise a common source of con- 
fusion to beginners. 

IV. THE XJRINE IN DISEASE 

In this section the characteristics of the urine in those 
diseases which produce distinctive urinary changes will 
be briefly reviewed. 

I. Renal Hyperemia. — Active hyperemia is usually an 
early stage of acute nephritis, but may occur independ- 
ently as a result of temporary irritation. The urine is 
generally decreased in quantity, highly colored, and 
strongly acid. Albumin is always present — usually in 
traces only, but sometimes in considerable amount for a 



174 



THE URINE 



day or two. The sediment contains a few hyaline and 
finely granular casts and an occasional red blood-cell. 




Fig. 63. — Sediment from acute hemorrhagic nephritis: Red hloo.l-corpuscles; leukocytes; 
renal cells not fattily degenerated; epithelial and blood-casts (Jakob). 




Fig. 64. — Sediment from chronic parenchymatous nephritis: Hyaline (with cells 
attached), waxy, brown granular, fatty, and epithelial casts; fattily degenerated renal cells, 
and a few white and red blood-corpuscles (Jakob). 

In very severe hyperemia the urine approaches that of 
acute nephritis. 

Passive hyperemia occurs most commonly in diseases of 



THE URINE IN DISEASE 175 

the heart and liver and in pregnancy. The quantity of 
urine is somewhat low and the color high, except in 
pregnancy. Albumin is present in small amount only. 
The sediment contains a very few hyaHne or finely 
granular casts. In pregnancy the amount of albumin 
should be carefully watched, as any considerable quan- 
tity, and especially a rapid increase, strongly suggests 
approaching eclampsia. 

2. Nephritis. — The various degenerative and inflam- 
matory conditions grouped under the name of nephritis 
have certain features in common. The urine in all 
cases contains albumin and tube-casts, and in all well- 
marked cases shows a decrease of normal solids, especially 
of urea and the chlorids. In chronic nephritis, especially 
of the interstitial type, there may be remissions during 
which the urine is practically normal. The character- 
istics of the different forms are well shown in the table 
on page 176, modified from Hill. 

3. Renal Tuberculosis. — The urine is pale, usually 
cloudy. The quantity may not be affected, but is apt to 
be increased. In early cases the reaction is faintly acid 
and there are traces of albumin and a few renal cells. 
In advanced cases the urine is alkaline, has an offensive 
odor, and is irritating to the bladder. Albumin in vary- 
ing amounts is always present. Pus is nearly always 
present, though frequently not abundant. It is generally 
intimately mixed with the urine, and does not settle so 
quickly as the pus of cystitis. Casts, though present, are 
rarely abundant, and are obscured by the pus. Small 
amounts of blood are common. Tubercle bacilli are 
nearly always present, although animal inoculation may 
be necessary to detect them. 



176 



THE URINE 



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THE URINE IN DISEASE 



177 



4. Renal Calculus. — The urine is usually somewhat 
concentrated, with high color and strongly acid reaction. 
Small amounts of albumin and a few casts may be pres- 
ent as a result of kidney irritation. Blood is frequently 
present, especially in the daytime and after severe ex- 
ercise. Crystals of the substance composing the cal- 
culus — uric acid, calcium oxalate, cystin — may often 
be found. The presence of a calculus generally produces 




Fig. 65. — Sediment from calculous pyelitis: Numerous pus-corpuscles, red blood-cor- 
puscles, and caudate and irregular epithelial cells; a combination of hyaline and pus- 
casts, and a few uric-acid crystals (Jakob). 



pyelitis, and variable amounts of pus then appear, the 
urine remaining acid in reaction. 

5. Pyelitis. — In pyelitis the urine is slightly acid, and 
contains a small or moderate amount of pus, together 
with many spindle and caudate epithelial cells. Pus- 
" casts may appear if the process extends up into the kid- 
ney tubules (see Fig. 65). Albumin is always present, 
and its amount, in proportion to the amount of pus, is 
decidedly greater than is found in cystitis. This fact is 
12 



178 



THE URINE 



of much value in differential diagnosis. Even when pus 
is scanty, albumin is rarely under 0.15 per cent., which 
is the maximum amount found in cystitis with abundant 
pus. 

6. Cystitis.— In acute and subacute cases the urine is 
acid and contains a variable amount of pus, with many 
epithelial cells from the bladder — chiefly large round, 
pyriform, and rounded squamous cells. Red blood- 
corpuscles are often numerous. 




Fig. 66. — Sediment from cystitis (chronic): Numerous pus-corpuscles, epithelial celb 
from the bladder, and bacteria; a few red blood-corpuscles and triple phosphate and 
ammonium urate crystals (Jakob). 

In chronic cases the urine is generally alkaline. It is 
pale and cloudy from the presence of pus, which is abun- 
dant and settles readily into a viscid sediment. The 
sediment usually contains abundant amorphous phos- 
phates and crystals of triple phosphate and ammonium 
urate. Vesical epithelium is common. Numerous bac- 
teria are always present (see Fig. 66). 

7. Vesical Calculus, Tumors, and Tuberculosis. — 
These conditions produce a chronic cystitis, with its 



THE URINE IN DISEASE 1 79 

characteristic urine. Blood, however, is more frequently 
present and more abundant than in ordinary cystitis. 
With neoplasms, especially, considerable hemorrhages 
are apt to occur. Particles of the tumor are sometimes 
passed with the urine. No diagnosis can be made from 
the presence of isolated tumor cells. In tuberculosis 
tubercle bacilli can generally be detected. 

8. Diabetes Insipidus. — Characteristic of this disease 
is the continued excretion of very large quantities of pale, 
watery urine, containing neither albumin nor sugar. 
The specific gravity varies between i.ooi and 1.005. 
The daily output of solids, especially urea, is increased. 

9. Diabetes Mellitus. — The quantity of urine is very 
large. The color is generally pale, while the specific 
gravity is nearly always high — 1.030 to 1.050, very rarely 
below 1.020. The presence of glucose is the essential 
feature of the disease. The amount of glucose is often 
very great, sometimes exceeding 8 per cent., while the 
total elimination may exceed 500 gm. in twenty-four 
hours. It may be absent temporarily. Acetone is gen- 
erally present in advanced cases. Diacetic and oxy- 
butyric acids may be present, and usually warrant an 
unfavorable prognosis. Accompanying the acidosis there 
is a corresponding increase in amount of ammonia. 



CHAPTER III 

THE BLOOD 

Preliminary Considerations. — The blood consists of 
a fluid of complicated and variable composition, the 
plasma, in which are suspended great numbers of micro- 
scopic structures: viz., red corpuscles, white corpuscles, 
blood-platelets, and blood-dust. 

Red corpuscles, or erythrocytes, appear as biconcave 
discs, red when viewed by reflected light or in thick layer, 
and straw colored when viewed by transmitted light or 
in thin layer. They give the blood its red color. They 
are cells which have been highly differentiated for the 
purpose of carrying oxygen from the lungs to the tissues. 
This is accomplished by means of an iron-bearing pro- 
tein, hemoglobin, which they contain. In the lungs 
hemoglobin forms a loose combination with oxgyen, 
which it readily gives up when it reaches the tissues. 
Normal erythrocytes do not contain nuclei. They are 
formed from preexisting nucleated cells in the bone- 
marrow. 

White corpuscles, or leukocytes, are less highly differ- 
entiated cells. There are several varieties. They all 
contain nuclei, and most of them contain granules which 
vary in size and staining properties. They are formed 
chiefly in the bone-marrow and lymphoid tissues. 

Blood- platelets, or blood- plaques , are colorless or slightly 
bluish, spheric or ovoid bodies, about one- third or one- 

180 



PRELIMINARY CONSIDERATIONS l8l 

half the diameter of an erythrocyte. Their structure, 
nature, and origin have not been definitely determined. 

The blood-dust of Miiller consists of fine granules which 
have vibratory motion. Little is known of them. It has 
been suggested that they are granules from disintegrated 
leukocytes. 

The total amount of blood is usually given as one- 
thirteenth of the body weight, but more recent investi- 
gations indicate that it averages about one-twentieth. 

The reaction is alkaline to litmus. 

The color is due to the presence of hemoglobin in the 
red corpuscles, the difference between the bright red of 
arterial blood and the purplish red of venous blood de- 
pending upon the relative proportions of oxygen and 
carbon dioxid. The depth of color depends upon the 
amount of hemoglobin. In very severe anemias the 
blood may be so pale as to be designated as " watery." 
The formation of carbon-monoxid-hemoglobin in coal- 
gas poisoning gives the blood a bright cherry-red color; 
while formation of methemoglobin in poisoning with 
potassium chlorate and certain other substances gives 
a chocolate color. 

Coagulation consists essentially in the transformation 
of fibrinogen, one of the proteins of the blood, into fibrin 
by means of a ferment derived from disintegration of the 
leukocytes. The presence of calcium salts is necessary 
to the process. The resulting coagulum is made up of a 
meshwork of fibrin fibrils with entangled corpuscles and 
plaques. The clear, straw-colored fluid which is left 
after separation of the coagulum is called blood-serum. 
Normally, coagulation takes place in two to eight min- . 
utes after the blood leaves the Vessels. It is frequently 



l82 



THE BLOOD 



desirable to determine the coagulation time. The 
simplest method is to place a drop of blood upon a per- 
fectly clean slide, and to draw a needle through it at half- 
minute intervals. When the clot is dragged along by the 
needle, coagulation has taken place. This method is 
probably sufficient for ordinary clinical work. For very 
accurate results the method of Russell and Brodie, as 
modified by Boggs, is recommended. The instrument 
is shown in Fig. 67. A drop of blood is placed upon the 
cone, which is then quickly inverted in the moist chamber. 




Fig. 67. — Boggs' coagulation instrument: A, moist chamber; R, glass cone; C, tube through 
which air is blown. 



By means of a rubber bulb puffs of air are blown against 
the blood at intervals, while the motion of the corpuscles 
is watched with a low-power objective. Coagulation is 
complete when the red cells move only en masse and 
spring back to their original position when the current 
ceases. Coagulation is notably delayed in hemophilia 
and icterus and after administration of citric acid. It 
is hastened by administration of calcium salts. 

For certain purposes, especially in bacteriologic and 
opsonic work, it is desirable to prevent coagulation 



PRELIMINARY CONSIDERATIONS 1 83 

of the blood that is withdrawn. This may be accom- 
plished by receiving it directly into a solution of i per 
cent, sodium citrate (or ammonium oxalate) and 0.85 
per cent, sodium chlorid. This precipitates the calcium 
salts which are necessary to coagulation. 

For most clinical examinations only one drop of blood 
is required. This may be obtained from the lobe of the 
ear, the palmar surface of the tip of the finger, or, in the 
case of infants, the plantar surface of the great toe. 
In general, the finger will be found most convenient. 
With nervous children the lobe of the ear is preferable, 
as it prevents their seeing what is being done. An ede- 
matous or congested part should be avoided. The site 




l-'ig. 68. — Daland's blood-lancet. 

should be well rubbed with alcohol to remove dirt and 
epithelial debris and to increase the amount of blood 
in the part. After allowing sufficient time for the circu- 
lation to equalize, the skin is punctured with a blood 
lancet (of which there are several patterns upon the 
market) or some substitute, as a Hagedorn needle, 
aspirating needle, trocar, or a pen with one of its nibs 
broken off. Nothing is more unsatisfactory than an 
ordinary sewing-needle. The lancet should be cleaned 
with alcohol before and after using, but need not be 
sterilized. It must be very sharp. If the puncture be 
made with a. firm, quick, rebounding stroke, it is practically 
painless. The first drop of blood which appears should 



184 THE BLOOD 

be wiped away, and the second used for examination. 
The blood should not be pressed out, since this dilutes 
it with serum from the tissues; but moderate pressure 
some distance above the puncture is allowable. 

When a larger amount of blood is required, it may be 
obtained with a sterile hypodermic S}Tinge from one of 
the veins at the elbow, as described on p. 245. 

CHnical study of the blood may be discussed under the 
following heads: I. Hemoglobin. II. Enumeration of 
erythrocytes. III. Color index. IV. Volume index. 
V. Enumeration of leukocytes. VI. Enumeration of 
plaques. VII. Study of stained blood. VIII. Blood 
parasites. IX. Serum reactions. X. Tests for recog- 
nition of blood. XL Special blood pathology. 

I. HEMOGLOBIN 

Hemoglobin is an iron-bearing protein. It is found 
only within the red corpuscles, and constitutes about 
90 per cent, of their weight. The actual amount of 
hemoglobin is never estimated clinically: it is the rela- 
tion which the amount present bears to the normal which 
is determined. Thus the expression, " 50 per cent, hemo- 
globin," when used clinically, means that the blood con- 
tains 50 per cent, of the normal. Theoretically, the 
normal would be 100 per cent., but with the methods of 
estimation in general use the blood of healthy persons 
ranges from 85 to 105 per cent.; these figures may, there- 
fore, be taken as normal. 

Increase of hemoglohin, or hyperchromemia, is un- 
common, and is probably more apparent than real. It 
accompanies an increase in number of erythrocytes, and 
may be noted in change of residence from a lower to a 



HEMOGLOBIN 1 85 

higher altitude ; in poorly compensated heart disease with 
cyanosis; in concentration of the blood from any cause, as 
the severe diarrhea of cholera, and in " idiopathic 
polycythemia." 

Decrease of hemoglobin, or oligochromemia, is very 
common and important. It is the most striking feature 
of the secondary anemias (p. 277). Here the hemo- 
globin loss may be slight or very great. In mild cases a 
slight decrease of hemoglobin is the only blood change 
noted. In very severe cases, especially in repeated 
hemorrhages, malignant disease, and infection by the 
hookworm and Dibothriocephalus latus, hemoglobin 
may fall to 15 per cent. Hemoglobin is always dimin- 
ished, and usually very greatly, in chlorosis, pernicious 
anemia, and leukemia. 

Estimation of hemoglobin is less tedious and usually 
more helpful than a red corpuscle count. It offers 
the simplest and most certain means of detecting the 
existence and degree of anemia, and of judging the effect 
of treatment in anemic conditions. Pallor, observed 
clinically, does not always denote anemia. 

There are many methods, but none is entirely satis- 
factory. Those which are most widely used are here 
described. 

(i) Von Fleischl Method. — ^The apparatus consists of a 
stand somewhat like the base and stage of a microscope 
(Fig. 69) . Under the stage is a movable bar of colored glass, 
shading from pale pink at one end to deep red at the other. 
The frame in which this bar is held is marked with a scale of 
hemoglobin percentages corresponding to the different shades 
of red. By means of a rack and pinion, the color-bar can be 
moved from end to end beneath a round opening in the center 



i86 



THE BLOOD 



of the stage. A small metal cylinder, which has a glass bot- 
tom and which is divided vertically into two equal compart- 
ments, can be placed over the opening in the stage so that one 
of its compartments lies directly over the color-bar. Accom- 
})anying the instrument are a number of short capillary tubes 
in metal handles. 

Having punctured the finger-tip or lobe of the ear, as al- 
ready described, wipe oflF the first drop of blood, and from the 




Fig. 69. — Von Fleischl's hcmoglobinomcter: a, Stand; h. narrow wedge-shaped piece 
of colored glass fitted into a frame ic), which passes under the chamber; d, hollow metal 
cylinder, divided into two compartments, which holds the blood and water; e. plaster-of- 
Paris plate from which the light is reflected through the chamber; /, screw by which the 
frame containing the graduated colored glass is moved; g, capillary tube to collect the 
blood; h, pipet for adding the water; «, opening through which may be seen the scale 
(ixlicating jiercentage of hemoglobin. 



second fill one of the capillary tubes. Hold the tube hori- 
zontally, and touch its tip to the drop of blood, which will 
readily flow into it if it be clean and dry. Avoid getting any 
blood upon its outer surface. With a medicine-dropper, rinse 
the blood from the tube into one of the compartments of the 
cylinder, using distilled water, and mix well. Fill both com- 



HEMOGLOBIN 1 87 

partments level full with distilled water, and place the cylin- 
der over the opening in the stage, so that the compartment 
which contains only water lies directly over the bar of colored 
glass. If there are any clots in the hemoglobin compart- 
ment, clean the instrument and begin again. 

In a dark room, with the light from a candle reflected up 
through the cylinder, move the color-bar along with a jerking 
motion until both compartments have the same depth of color. 
The number upon the scale corresponding to the portion of the 
color-bar which is now under the cylinder gives the percentage 
of hemoglobin. While comparing the two colors, place the 
instrument so that they will fall upon the right and left halves 
of the retina, rather than upon the upper and lower halves; 
and protect the eye from the light with a cylinder of paper or 
pasteboard. After use, clean the metal cylinder with water, 
and wash the capillary tube with water, alcohol, and ether, 
successively. Results with this instrument are accurate to 
within about 5 per cent. 

A recent modification of the von Fleischl apparatus by 
Miescher gives an error which need not exceed i per cent. 
It is, however, better adapted to laboratory use than to the 
needs of the practitioner. 

(2) The Sahli hemoglobinometer (Fig. 70) is an improved 
form of the well-known Gowers instrument. It consists of a 
hermetically sealed comparison tube containing a i per cent, 
solution of acid hematin, a graduated test-tube of the same 
diameter, and a pipet of 20-c.mm. capacity. The two tubes 
are held in a black frame with a white ground-glass back. 

Place a few drops of decinormal hydrochloric acid solution 
in the graduated tube. Obtain a drop of blood and draw it 
"into the pipet to the 20 c.mm. mark. Wipe off the tip of the 
pipet, blow its contents into the hydrochloric acid solution in 
the tube, and rinse well. In a few minutes the hemoglobin is 
changed to acid hematin. Place the two tubes in the com- 
partments of the frame, and dilute the fluid with water drop 



i88 



THE BLOOD 



by drop, mixing after each addition, until it has exactly the 
same color as the comparison tube. The graduation corre- 
sponding to the surface of the fluid then indicates the per- 
centage of hemoglobin. Decinormal hydrochloric acid solu- 
tion may be prepared with suflficient accuracy for this purpose 




Fig. 70. — Sahli's hemoglobinometer. 



by adding 15 c.c. of the concentrated acid to 985 c.c. distilled 
water. A little chloroform should be added as a preservative. 
This method is very satisfactory in practice, and is accurate 
to within 5 per cent. The comparison tube is said to keep its 
color indefinitely, but, unfortunately, not all the instruments 
upon the market are well standardized. 



HEMOGLOBIN 1 89 

(3) Dare's hemoglobinometer (Fig. 71) differs from the 
others in using undiluted blood. The blood is allowed to flow 
by capillarity into the slit between two small plates of glass. 
It is then placed in the instrument and compared with differ- 
ent portions of a circular disc of colored glass. The reading 
must be made quickly, before clotting takes place. This 
instrument is easy to use, and is one of the most accurate. 



Fig. 71. — Dare's hemoglobinometer. 

(4) Hammerschlag Method. — This is an indirect method 
which depends upon the fact that the percentage of hemo- 
globin varies directly with the specific gravity of the blood. 
It yields fairly accurate results except in leukemia, where the 
large number of leukocytes disturbs the relation, and in 
dropsical conditions. 

Mix chloroform and benzol in a urinometer tube, so that 
the specific gravity of the mixture is near the probable specific 
gravity of the blood. Add a drop of blood by means of a 
pipet of small caliber. A pipet hke that shown in Fig. 161, A 
will be found satisfactory. If the drop floats near the surface, 
add a little benzol ; if it sinks to the bottom, add a little chloro- 
form. When it remains stationary near the middle, the mix- 



IQO 



THE BLOOD 



ture has the same specific gravity as the blood. Take the 
specific gravity with a urinometer, and obtain the correspond- 
ing percentage of hemoglobin from the following table: 



Gravity. 



Hemoglobin 
Per Cent. 



Specific 
Gravity. 



Hemoglobin 
Per Cent- 



1.033-1.035 25-30 1.048-1.050 s5-^5 



1.035-1.038 30-35 

1.038-1.040 35-40 

1.040-1.045 40-45 

1.045-1.048 45-55 



1. 050-1.053 65-70 

1.053-1.055 70-75 

1.055-1.057 75-85 

1.057-1.060 85-95 



For accurate results with this method, care and patience 
are demanded. The following precautions must be observed: 




Fig. 72. — Tallquist's bemoglobin scale. 



(a) The two fluids must be well mixed after each addition 
of chloroform or benzol. Close the tube with the thumb and 
invert several times. Should this cause the drop of blood to 



HEMOGLOBIN I9I 

break up into very small ones, adjust the specific gravity as 
accurately as possible with these, and test it with a fresh drop. 

(b) The drop of blood must not be too large; it must not 
contain an air-bubble, it must not adhere to the side of the 
tube, and it must not remain long in the fluid. 

(c) The urinometer must be standardized for the chloro- 
form-benzol mixture. Most urinometers give a reading two 
or three degrees too high, owing to the low surface tension. 
Make a mbcture such that a drop of distilled water will re- 
main suspended in it {i. e., with a specific gravity of i.ooo) 
and correct the urinometer by this. 

(5) Tallquist Method. — The popular Tallquist hemo- 
globinometer consists simply of a book of small sheets of ab- 
sorbent paper and a carefully printed scale of colors (Fig. 72). 

Take up a large drop of blood with the absorbent paper, 
and when the humid gloss is leaving, before the air has dark- 
ened the hemoglobin, compare the stain with the color 
scale. The color which it matches gives the percentage 
of hemoglobin. Except in practised hands, this method is 
accurate only to within 10 or 20 per cent. 

Of the methods given, the physician should select the 
one which best meets his needs. With any method, 
practice is essential to accuracy. The von Fleischl has 
long been the standard instrument, but has lately fallen 
into some disfavor. For accurate work the best instru- 
ments are the von Fleischl-Miescher and the Dare. They 
are, however, expensive, and it is doubtful whether they 
are enough more accurate than the Sahli instrument to 
justify the difference in cost. The latter is probably the 
most satisfactory for the practitioner, provided a well- 
standardized color- tube is obtained. The specific gravity 
method is very useful when special instruments are not 
at hand. The Tallquist scale is so inexpensive and so 



192 THE BLOOD 

convenient that it should be used by every physician at 
the bedside and in hurried office work ; but it should not 
supersede the more accurate methods. 

II. ENUMERATION OF ERYTHROCYTES 

In health there are about 5,000,000 red corpuscles per 
cubic millimeter of blood. Normal variations are shght. 
The number is generally a little less — about 4,500,000 — in 
women. 

Increase of red corpuscles, or polycythemia, is unimpor- 
tant. There is a decided increase following change of 
residence from a lower to a higher altitude, averaging 
about 50,000 corpuscles for each 1000 feet, but frequently 
much greater. The increase, however, is not permanent. 
In a few months the erythrocytes return to nearly their 
original number. Three views are ofTered in explanation : 
(a) Concentration of the blood, owing to increased evap- 
oration from the skin; (b) stagnation of corpuscles in the 
peripheral vessels because of lowered blood-pressure; 
(c) new formation of corpuscles, this giving a compensa- 
tory increase of aeration surface. 

Pathologically, polycythemia is uncommon. It may 
occur in: (a) Concentration of the blood from severe 
watery diarrhea; (b) chronic heart disease, especially the 
congenital variety, with poor compensation and .cyanosis; 
and (c) idiopathic polycythemia, which is considered to be 
an independent disease, and is characterized by cyanosis, 
blood counts of 7,000.000 to 10,000,000, hemoglobin 120 
to 150 per cent., and a normal number of leukocytes. 

Decrease of red corpuscles, or oligocythemia. Red 
corpuscles and hemoglobin are commonly decreased 
together, although usually not to the same extent. 



ENUMERATION OF ERYTHROCYTES 



195 



Oligocythemia occurs in all but the mildest symp- 
tomatic anemias. The blood-count varies from near the 
normal in moderate cases down to 1,500,000 in very 
severe cases. There is always a decrease of red cells in 
chlorosis, but it is often slight, and is relatively less than 
the decrease of hemoglobin. Leukemia gives a decided 
oligocythemia, the average count being about 3,000,000. 
The greatest loss of red cells occurs in pernicious anemia, 
where counts below 1,000,000 are not uncommon. 



OlOOmm 


H 






73. — Thoma-Zeiss heraocytometer: a. Slide used in counting; h, sectional view; 
d, red pipet; e, white pipet. 

The most widely used and most satisfactory instru- 
ment for counting the corpuscles is that of Thoma-Zeiss, 
The hematocrit is not to be recommended for accuracy, 
since in anemia, where blood-counts are most important, 
the red cells vary greatly in size and probably also in 
elasticity. The hematocrit is, however, useful in de- 
termining the relative volume of corpuscles and plasma 
(Volume Index, p. 200), and seems to be gaining in favor. 

13 



194 



THE BLOOD 



The Thoma-Zeiss instrument consists of two pipets 
for diluting the blood and a counting chamber (Fig. 73). 
The counting chamber is a glass slide with a square platform 
in the middle. In the center of the platform is a circular 
opening, in which is set a small circular disc in such a manner 
that it is surrounded by a " ditch," and that its surface is 



Fig. 74. — Ordinary ruling of counting chamber, showing red corpuscles in left 



upper 



exactly one-tenth of a millimeter below the surface of the 
square platform. Upon this disc is ruled a square millimeter, 
subdivided into 400 small squares. Each fifth row of small 
squares has double rulings for convenience in counting (Fig. 
74). A thick cover-glass, ground perfectly plane, accompa- 
nies the counting chamber. Ordinary cover-glasses are of 
uneven surface, and should not be used with this instrument. 



ENUMERATION OF ERYTHROCYTES 1 95 

It is evident that, when the cover-glass is in place upon 
the platform, there is a space exactly one-tenth of a millimeter 
thick between it and the disc; and that, therefore, the square 
millimeter ruled upon the disc forms the base of a space 
holding exactly one-tenth of a cubic millimeter. 

Technic. — To count the red corpuscles, use the pipet with 
loi engraved above the bulb. It must be clean and dry. 
Obtain a drop of blood as already described. Suck blood 




Fig. 75. — Method of drawing blood into the pipet (Boston), 

into the pipet to the mark 0.5 or i. Should the blood go 
beyond the mark, draw it back by touching the tip of the pipet 
to a moistened handkerchief. Quickly wipe off the blood 
adhering to the tip, plunge it into the diluting fluid, and suck 
the fluid up to the mark loi, slightly rotating the pipet 
meanwhile. This dilutes the blood i : 200 or i : 100, accord- 
ing to the amount of blood taken. Except in cases of severe 
anemia, a dilution of i : 200 is preferable. Close the ends 



196 THE BLOOD 

of the pipet with the fingers, and shake vigorously until the 
blood and diluting fluid are well mixed. 

When it is not convenient to count the corpuscles at once, 
place a heavy rubber band around the pipet so as to close 
the ends, inserting a small piece of rubber-cloth or other 
tough, non-absorbent material, if necessary, to prevent the tip 
from punching through the rubber. It may be kept thus for 
twenty-four hours or longer. 

When ready to make the count, clean the counting 
chamber and cover-glass, and place a sheet of paper over 
them to keep off dust. IVIix the fluid thoroughly by shak- 
ing; blow two or three drops from the pipet, wipe of! its 
tip, and then place a small drop (the proper size can be 
learned only by experience) upon the disc of the counting 
chamber. Adjust the cover immediately. Hold it by diag- 
onal corners above the drop of fluid so that a third corner 
touches the slide and rests upon the edge of the platform. 
Place a finger upon this corner, and, by raising the finger, 
allow the cover to fall quickly into place. If the cover be 
properly adjusted, faint concentric lines of the prismatic 
colors — Newton's rings — can be seen between it and the plat- 
form when the slide is viewed obliquely. They indicate that 
the two surfaces are in close apposition. If they do not ap- 
pear at once, slight pressure upon the cover may bring them 
out. Failure to obtain them is usually due to dirty slide or 
cover — both must be perfectly clean and free from dust. 
The drop placed upon the disc must be of such size that, when 
the cover is adjusted, it nearly or quite covers the disc, and 
that none of it runs over into the " ditch." There should 
be no bubbles upon the ruled area. 

Allow the corpuscles to settle for a few minutes, and then 
examine with a low power to see that they are evenly dis- 
tributed. If they are not evenly distributed over the whole disc. 
the counting chamber must be cleaned and a new drop placed 
in it. 



ENUMERATION OF ERYTHROCYTES 



197 



Probably the most satisfactory objective for counting is the 
special 4 mm. with long working distance. To understand 
the principle of counting, it is necessary to remember that 
the large square (400 small squares) represents a capacity 
of one-tenth of a cubic millimeter. Find the number of 
corpuscles in the large square, multiply by 10 to find the 




Fig. 76. — Appearance of microscopic field in counting red corpuscles. The arrow indicates 
the squares to be counted. 



number in i c.mm. of the diluted blood, and finally, by the 
dilution, to find the number in i c.mm. of undiluted blood. 
. Instead of actually counting all the corpuscles, it is customary 
to count those in only a limited number of small squares, 
and from this to calculate the number in the large square. 
Nearly every worker has his own method of doing this. 
The essential thing is to adopt a method and adhere to it. 



198 THE BLOOD 

In practice a convenient procedure is as follows: With a 
dilution of i : 200, count the cells in 80 small squares, and to 
the sum add 4 ciphers; with dilution of i : 100, count 40 small 
squares and add 4 ciphers. Thus, if with i : 200 dilution, 450 
corpuscles were counted in 80 squares, the total count would 
be 4,500,000 per c.mm. This method is sufficiently accurate 
for all clinical purposes, provided the corpuscles are evenly 
distributed and three drops from the pipet be counted. It is 
convenient to count a block of 20 small squares, as indicated 
in Fig. 76, in each corner of the large square. Four columns 
of 5 squares each are counted. The double rulings show when 
the bottom of a column has been reached and also indicate 
the fourth column. In the writer's opinion it is easier to 
count in vertical than horizontal rows. If distribution be 
even, the difference between the number of cells in any two 
such blocks should not exceed twenty. In order to avoid 
confusion in counting cells which lie upon the border-lines, 
the following rule is generally adopted: Corpuscles which 
touch the upper and left sides should be counted as if within the 
squares, those touching the lower and right sides, as outside; and 
vice versd.. 

Diluting Fluids. — The most widely used are Hayem's and 
Toisson's. Both of these have high specific gravities, so that, 
when well mixed, the corpuscles do not separate quickly. 
Toisson's fluid is probably the better for beginners, because it 
is colored and can easily be seen as it is drawn into the pipet. 
It stains the nuclei of leukocytes blue, but this is no real ad- 
vantage. It must be filtered frequently. 

Hayem's Fluid. Toisson's Fturo. 

Mercuric chlorid 0.5 Methyl-violet, 5 B 0.025 

Sodium sulphate 5.0 Sodium chlorid i.ooo 

Sodium chlorid i.o Sodium sulphate 8.000 

Distilled water 200.0 Glycerin 30.000 

Distilled water 160.000 



ENUMERATION OF ERYTHROCYTES 1 99 

Sources of Error. — The most common sources of error in 
making a blood count are: 

(a) Inaccurate dilution, either from faulty technic or 
inaccurately graduated pipets. The instruments made by 
Zeiss can be relied upon. 

(b) Too slow manipulation, allowing a little of the blood to 
coagulate and remain in the capillary portion of the pipet, 

(c) Inaccuracy in depth of counting chamber, which some- 
times results from softening of the cement by alcohol or heat. 
The slide should not be cleaned with alcohol nor left to lie 
in the warm sunshine. 

(d) Uneven distribution of the corpuscles. This results 
when the blood is not thoroughly mixed with the diluting 
fluid, or when the cover-glass is not applied soon enough after 
the drop is placed upon the disc. 

Cleaning the Instrument. — The instrument should be 
cleaned immediately after using, and the counting chamber 
and cover must be cleaned again just before use. 

Draw through the pipet, successively, water, alcohol, ether, 
and air. This can be done with the mouth, but it is much 
better to use a rubber bulb or suction filter pump. When the 
mouth is used, the moisture of the breath will condense upon 
the interior of the pipet unless the fluids be shaken and not 
blown out. If blood has coagulated in the pipet — which hap- 
pens when the work is done too slowly — dislodge the clot with 
a horsehair, and clean with strong sulphuric acid, or let the 
pipet stand over night in a test-tube of the acid. Even if the 
pipet does not become clogged, it should be occasionally 
cleaned in this way. When the etched graduations on the 
pipets become dim, they can be renewed by rubbing with a 
grease pencil. 

Wash the counting-chamber and the cover with water and 
dry them with clean soft linen. Alcohol may be used to clean 
the latter, but never the former. 



200 THE BLOOD 

III. COLOR INDEX 

This is an expression which indicates the amount of 
hemoglobin in each red corpuscle compared with the 
normal amount. For example, a color index of i.o 
indicates that each corpuscle contains the normal amount 
of hemoglobin; of 0.5, that each contains one-half the 
normal. 

The color index is most significant in chlorosis and 
pernicious anemia. In the former it is usually much 
decreased; in the latter, generally much increased. In 
symptomatic anemia it is generally moderately dimin- 
ished. 

To obtain the color index, divide the percentage of hemo- 
globin by the percentage of corpuscles. The percentage of 
corpuscles is found by multiplying the first two figures of the 
red corpuscle count by two. This simple method holds good 
for all counts of 1,000,000 or more. Thus, a count of 2,500,000 
is 50 per cent, of the normal. If, then, the hemoglobin has 
been estimated at 40 per cent., divide 40 (the percentage of 
hemoglobin) by 50 (the percentage of corpuscles). This 
gives i, or 0.8, as the color index. 

IV. VOLUME INDEX 

The term " volume index " was introduced by Capps 
to express the average size of the red cells of an individual 
compared with their normal size. It is the quotient 
obtained by dividing the volume of red corpuscles (ex- 
pressed in percentage of the normal) by the number of 
red corpuscles, also expressed in percentage of the nor- 
mal. 

The volume index more or less closely parallels the 
color index, and variations have much the same sig- 



^m^ rr-r rr- ^^^ 



VOLUME 

r. u ■, r r^ ' J '^1 /^ I 

mficance. The following are avetages- 6^ ttr^ examma- 

tions reported by Larrabee in the Journal of Medical ^ 

Research: 

Red corpuscles Hemoglobin per _ , , , , 

, . ^ u c Li- Color Volume 

per cubic cent by Sahli . . 

..... . . . mdez. mdex. 

millimeter. instrument. 

Normal males 5,267,250 103.0 0.98 1.007 

Normal females 4,968,667 106.0 1.06 i.ooi 

Primary pernicious anemia . 1,712,166 50.0 1.47 1.270 

Secondary anemia 3, 737, 160 61.0 0.81 0.790 

Chlorosis 3,205,000 34.5 0.55 0.695 

Method. — The red cells are counted and the percentage of 
red cells calculated as for the color index. 

The volume percentage is obtained with the hematocrit 
as follows: Fill the hematocrit tubes (Fig. 77) with blood, and 
before coagulation takes place insert them in the frame and 
centrifugalize for three minutes at about 8000 to 10,000 
revolutions a minute. The red cells collect at the bottom 
and, normally, make up one-half of the total column of blood. 



{ "10' • ■ £;^''j^Q- ; ;j^' ; rj^< ' ^^''^' q'q' g ^ 




Fig. 77. — Daland hematocrit for use with the centrifuge. 

Multiply the height of the layer of red cells (as indicated by 
the graduations upon the side of the tube) by 2 to obtain the 
volume percentage. When the examination cannot be made 
immediately after the blood is obtained, the method of 
Larrabee is available. This consists in mixing a trace of so- 
dium oxalate with a few drops of blood to prevent coagulation, 
drawing this mixture into a tube of about 2-mm. caliber and 
waiting until sedimentation is complete — usually about three 
days. The height of the column is then measured with a 



202 XMK BLOOD 

miUimetpr saiJe and tlie percentage relation to the normal 
calculated. 

After the volume of the red cells and the red corpuscle 
count are thus expressed in percentages, divide the former 
by the latter to find the volume index. Example: Suppose 
the volume percentage is 80 (the reds reaching to mark 40 on 
hematocrit tube) and that the red count is 50 per cent, of 
the normal (2,500,000 per c.mm.), then |^ or 1.6 is the vol- 
ume index. 

V. ENUMERATION OF LEUKOCYTES 

The normal number of leukocytes varies from 5000 to 
10,000 per cubic millimeter of blood. The number is 
larger in robust individuals than in poorly nourished 
ones, and if disease be excluded, may be taken as a 
rough index of the individual's nutrition. Since it is 
well to have a definite standard, 7500 is generally adopted 
as the normal for the adult. With children the number 
is somewhat greater, counts of 12,000 and 15,000 being 
common in healthy children under twelve years of age. 

Decrease in Number of Leukocytes 

Decrease in number of leukocytes, or leukopenia, is not 
important. It is common in persons who are poorly 
nourished, although not actually sick. The infectious 
diseases in which leukocytosis is absent (p. 206) often 
cause a slight decrease of leukocytes. Chlorosis may 
produce leukopenia, as also pernicious anemia, which 
usually gives it in contrast to the secondary anemias, 
which are frequently accompanied by leukocytosis. 
Leukocyte counts are, therefore, of some aid in the dififer- 
ential diagnosis of these conditions. 



enumeration of leukocytes 203 

Increase in Number of Leukocytes 
Increase in number of leukocytes is common and of 
great importance. It may be considered under two 
heads: 

A. Increase of leukocytes due to chemotaxis and 
stimulation of the blood-making organs, or leukocytosis. 
The increase affects one or more of the normal varieties. 

B. Increase of leukocytes due to leukemia. Normal 
varieties are increased, but the characteristic feature is 
the appearance of great numbers of abnormal cells. 

The former may be classed as a transient, the latter, as 
a permanent, increase. 

A. Leukocytosis 

This term is variously used. By some it is applied to 
any increase in number of leukocytes; by others it is 
restricted to increase of the polymorphonuclear neutro- 
philic variety. As has been indicated, it is here taken 
to mean a transient increase in number of leukocytes, 
that is, one caused by chemotaxis and stimulation of the 
blood-producing structures, in contrast to the permanent 
increase caused by leukemia. 

By chemotaxis is meant that property of certain agents 
by which they attract or repel living cells — positive 
chemotaxis and negative chemotaxis respectively. An 
excellent illustration is the accumulation of leukocytes 
at the site of inflammation, owing to the positively 
chemotactic influence of bacteria and their products. A 
great many agents possess the power of attracting leuko- 
cytes into the general circulation. Among these are 
many bacteria and certain organic and inorganic poisons. 

Chemotaxis alone will not explain the continuance of 



204 THE BLOOD 

leukocytosis for more than a short time. It is probable 
that substances which are positively chemotactic also 
stimulate the blood-producing organs to increased forma- 
tion of leukocytes; and in at least one form of leukocytosis 
such stimulation apparently plays the chief part. 

As will be seen later, there are several varieties of leu- 
kocytes in normal blood, and most chemotactic agents 
attract only one variety, and either repel or do not in- 
fluence the others. It practically never happens that 
all are increased in the same proportion. The most 
satisfactory classification of leukocytoses is, therefore, 
based upon the type of leukocyte chiefly affected. 

Theoretically, there should be a subdivision for each 
variety of leukocyte, e. g., polymorphonuclear leuko- 
cytosis, lymphocyte leukocytosis, eosinophilic leuko- 
cytosis, large mononuclear leukocytosis, etc. Practi- 
cally, however, only two of these, polymorphonuclear 
leukocytosis and lymphocyte leukocytosis, need be con- 
sidered under the head of Leukocytosis. Increase in 
number of the other leukocytes will be considered 
when the individual cells are described (pp. 230-243). 
They are present in the blood in such small numbers 
normally that even a marked increase scarcely afi^ects 
the total leukocyte count; and, besides, substances 
which attract them into the circulation frequently repel 
the pol>'morphonuclears, so that the total number of 
leukocytes may actually be decreased. 

The polymorphonuclear neutrophils are capable of 
active ameboid motion, and are by far the most numerous 
of the leukocytes. Ljonphocytes are about one-third 
as numerous and have little independent motion. As 
one would, therefore, expect, marked differences exist 



ENUMERATION OF LEUKOCYTES 205 

between the two types of leukocytosis: polynuclear 
leukocytosis is more or less acute, coming on quickly and 
often reaching high degree; whereas lymphocyte leuko- 
cytosis is more chronic, comes on more slowly, and is 
never so marked. 

I. Polymorphonuclear Neutrophilic Leukocytosis. — 
Polymorphonuclear leukocytosis may be either physi- 
ologic or pathologic. A count of 20,000 would be con- 
sidered a marked leukocytosis; of 30,000, high; above 
50,000, extremely high. 

(i) Physiologic Polymorphonuclear Leukocytosis. — 
This is never very marked, the count rarely exceeding 
15,000 per cubic millimeter. It occurs: (a) In the new- 
born; (b) in pregnancy; (c) during digestion, and (d) 
after cold baths. There is moderate leukocytosis in the 
moribund state: this is commonly classed as physiologic, 
but is probably due mainly to temiinal infection. 

The increase in these conditions is not limited to the 
polymorphonuclears. Lymphocytes are likewise in- 
creased in varying degrees, most markedly in the new- 
bom. 

In view of the leukocytosis of digestion, the hour 
at which a leukocyte count is made should always be 
recorded. Digestive leukocytosis is most marked three 
to five hours after a hearty meal rich in protein. It is 
absent in pregnancy and when leukocytosis from any 
other cause exists. It is usually absent in cancer of the 
stomach, a fact which may be of some help in the diag- 
nosis of this condition, but repeated examinations are 
essential. 

(2) Pathologic Polymorphonuclear Leukocytosis. — 
In general, the response of the leukocytes to chemotaxis 



2o6 THE BLOOD 

is a conservative process. It has been compared to the 
gathering of soldiers to destroy an invader. This is 
accomplished partly by means of phagocytosis — actual 
ingestion of the enemy — and partly by means of chemic 
substances which the leukocytes produce. 

In those diseases in which leukocytosis is the rule the 
degree of leukocytosis depends upon two factors: the 
severity of the infection and the resistance of the individual. 
A well-marked leukocytosis usually indicates good resist- 
ance. A mild degree means that the body is not react- 
ing well, or else that the infection is too slight to call 
forth much resistance. Leukocytosis may be absent 
altogether when the infection is extremely mild, or when 
it is so severe as to overwhelm the organism before it can 
react. When leukocytosis is marked, a sudden fall in 
the count may be the first warning of a fatal issue. 
These facts are especially true of pneumonia, diphtheria, 
and abdominal inflammations, in which conditions the 
degree of leukocytosis is of considerable prognostic value. 

The classification here given follows Cabot: 

{a) Infections and Inflammatory. — The majority of 
infectious diseases produce leukocytosis. The most not- 
able exceptions are influenza, malaria, measles, tuber- 
culosis, except when invading the serous cavities or 
when complicated by mixed infection, and typhoid fever, 
in which leukocytosis indicates an inflammatory com- 
plication. 

All inflammatory and suppurative diseases cause leu- 
kocytosis, except when slight or well walled off'. Appen- 
dicitis has been studied with especial care in this connec- 
tion, and the conclusions now generally accepted prob- 
ably hold good for most acute intra-abdominal inflam- 



ENUMERATION OF LEUKOCYTES 207 

mations. A marked leukocytosis (20,000 or more) 
nearly always indicates abscess, peritonitis, or gan- 
grene, even though the clinical signs be slight. Absence 
of or mild leukocytosis indicates a mild process, or else 
an overwhelmingly severe one ; and operation may safely 
be postponed unless the abdominal signs are very marked. 
On the other hand, no matter how low the count, an in- 
creasing leukocytosis — counts being made hourly — indi- 
cates a spreading process and demands operation, regard- 
less of other symptoms. 

Leukocyte counts alone are often disappointing, but are 
of much more value when considered in connection with 
a diferential count of polymorphonuclears. (See p. 236.) 

{b) Malignant Disease. — Leukocytosis occurs in about 
one-half of the cases of malignant disease. In many 
instances it is probably independent of any secondary 
infection, since it occurs in both ulcerative and non- 
ulcerative cases. It seems to be more common in 
sarcoma than in carcinoma. Very large counts are rarely 
noted. 

{c) Posthemorrhagic. — Moderate leukocytosis follows 
hemorrhage and disappears in a few days. 

{d) Toxic. — This is a rather obscure class, which in- 
cludes gout, chronic nephritis, acute yellow atrophy 
of the liver, ptomain-poisoning, prolonged chloroform 
narcosis, and quinin-poisoning. Leukocytosis may or 
may not occur in these conditions, and is not important. 

(e) Drugs. — This also is an unimportant class. Most 
tonics and stomachics and many other drugs produce a 
slight leukoc3^tosis. 

2. Lymphocyte Leukocytosis.— This is characterized 
by an increase in the total leukocyte count, accom- 



2o8 THE BLOOD 

panied by an increase in the percentage of lymphocytes. 
The word " lymphocytosis " is often used in the same 
sense. It is better, however, to use the latter as refer- 
ring to any increase in the absolute number of lympho- 
cytes, without regard to the total count, since an ab- 
solute increase in number of lymphocytes is frequently 
accompanied by a normal or subnormal leukocyte count, 
owing to loss of polymorphonuclears. 

Non-phagocytic leukocytosis is probably due more to 
stimulation of blood-making organs than to chemotaxis. 
It is less common, and is rarely so marked as a poly- 
morphonuclear leukocytosis. When marked, the blood 
cannot be distinguished from that of lymphatic leukemia. 

A marked lymphocyte leukocytosis occurs in pertussis, 
and is of value in diagnosis. It appears early in the 
catarrhal stage, and persists until after convalescence. 
The average leukocyte count is about 17,000, lympho- 
cytes predominating. There is moderate lymphocyte 
leukocytosis in other diseases of childhood, as rickets, 
scurvy, and especially hereditary syphilis, where the 
blood-picture may approach that of pertussis. It 
must be borne in mind in this connection that lympho- 
cytes are normally more abundant in the blood of children 
than in that of adults. 

Slight lymphocyte leukocytosis occurs in many other 
pathologic conditions, but is of little significance. 

B. Leukemia 
This is an idiopathic disease of the blood-making 
organs, which is accompanied by an enormous increase 
in number of leukocytes. The leukocyte count some- 
times reaches 1,000,000 per cubic millimeter, and leu- 



ENUMERATION OF LEUKOCYTES 209 

kemia is always to be suspected when it exceeds 50,000. 
Lower counts do not, however, exclude it. The subject 
is more fully discussed later (p. 280). 

Method of Counting Leukocytes 
The leukocytes are counted with the Thoma-Zeiss 
instrument, already described. Recently, several new 
rulings of the disc have been introduced, notably the 
Zappert and the Tiirck (Fig. 79), which give a ruled 
area of nine square millimeters. They were devised for 
counting the leukocytes in the same specimen with the 
red corpuscles. The red ceUs are counted in the usual 
manner, after which all the leukocytes in the whole area 
of nine square millimeters are counted; and the number 
in a cubic millimeter of undiluted blood is then easily 
calculated. Leukocytes are easily distinguished from 
red cells, especially when Toisson's diluting fluid is used. 
This method may be used with the ordinary ruling by 
adjusting the microscopic field to a definite size, and 
counting a sufficient number of fields, as described later. 
Although less convenient, it is more accurate to count the 
leukocytes separately, with less dilution of the blood, as 
follows : 

Technic. — A larger drop of blood is required than for 
counting the erythrocytes, and more care in filling the pipet. 
Boggs has suggested a device (Fig. 78) which enables one to 
draw in the blood more slowly and hence more accurately. 
He cuts the rubber tube and inserts a Wright " throttle." 
This consists of a section of glass tubing in which a capillary 
tube drawn out to a fine thread is cemented with sealing wax. 
After sealing in place the tip is broken off with forceps, so 
that upon gentle suction it will just allow air to pass. 

14 



2IO 



THE BLOOD 



Use the pipet with 1 1 engraved above the bulb. Suck the 
blood to the mark 0.5 or i.o, and the diluting fluid to the 
mark 11. This gives a dilution of i : 20 or 1:10, respectively. 
The dilution of i : 20 is easier to make. Mix well by shaking 
in all directions except in the long axis of the pipet; blow out 
two or three drops, place a drop in the counting chamber, 
and adjust the cover as already described (p. 196). 




Fig. 78. — Boggs' " throttle control " for blood-counting pip>et, and enlarged diagram show- 
ing construction of the throttle. 



Examine with a low power to see that the cells are evenly 
distributed. Count with the 16 mm. objective and a high 
eye-piece, or with the long-focus 4 mm. and a low eye-piece. 
An 8 mm. objective will be found very satisfactory for this 
purpose. As one gains experience one will rely more upon 
the lower powers. 

With the ordinary ruling of the disc, count all the leuko- 
cytes in the large square, multiply by 10 to find the number in 



ENUMERATION OF LEUKOCYTES 



211 



I c.mm. of diluted blood, and by the dilution to find the 
number per c.mm. of undiluted blood. In every case at least 
200 leukocytes must be counted as a basis for calculation, and 
it is much better to coimt 500. This will necessitate exam- 
ination of several drops from the pipet. With the Zappert 



Fig. 79. — Turck nxling of counting chamber. 



and Tiirck rulings a sufficient number can usually be counted 
in one drop, but the opportunity for error is very much greater 
when only one drop is examined. 

In routine work the author's modification of the "circle " 
method is very satisfactory. It requires a 4 mm. objective, 



212 



THE BLOOD 



and is, therefore, especially desirable for beginners, who are 
usually unable accurately to identify leukocytes with a lower 
power. The student is frequently confused by particles of 
dirt, remains of red cells, and yeast cells which sometimes 
grow in the diluting fluid. Draw out the sliding tube of the 



1 1 

1 > 

--r r 

. 4. 1 


,___ 


r - 


1 


r 




1 ■ 1 r 
ill 1 

r r- ""--T r-- 

L 1 1 1 


1 1 

--T -t- 

1 1 


1 

1 t- - 

1 


1 

r -+ 

1 


r — 


1 1 ! ' ' 
r---T---T--,--;---;-- 

1 r , 1 


1 
- - -t - 

r -t- - 












hs 


i . 1 ; 

I. - 4^ 1- L I 












\ 


s^ 1 

\ 1 




/ 














\ 1 


■ / 

1 / L 
















\ \ ; 


1 


















1 \ 














( 


'y.iV-'X:. 


1 1 \ 
- 1 1 \ 
















-: 1 1-- 


1 1 \ 
1 1 

--4- ^---1 

1 1 

1 1 
__4- 1--..1 

1 1 

1 1 

1 1 

Fig. 8o. — Size o 


^, 










y 


V 


field re 


- -1 
■ - 
quii 


ed i 


, 1 

. ^ 

n count 


\- ■ 

ng leuk 


ocytes a 


^---l-r-^---|- 
1 : . ! 

s described in the text. 



microscope until the field of vision is such as shown in Fig. 
8o. One side of the field is tangent to one of the ruled lines, 
A, while the opposite side just cuts the comers, B and C, of 
the seventh squares in the rows above and below the diameter 
line. When once adjusted, a scratch is made upon the draw- 



ENUMERATION OF BLOOD-PLAQUES 213 

tube, SO that for future counts the tube has only to be drawn 
out to the mark. The area of this microscopic field is one- 
tenth of a square millimeter. With a dilution of i : 20, count 
the leukocytes in 20 such fields upon different parts of the disc 
without regard to the ruled lines, and to their sum add two 
ciphers. With dilution of i : 10, count 10 such fields, and 
add two ciphers. Thus, with i : 10 dilution, if 150 leukocytes 
were counted in 10 fields, the leukocyte count would be 15,000 
per c.mm. To compensate for possible unevenness of dis- 
tribution, it is best to count a row of fields horizontally and a 
row vertically across the disc. This method is applicable 
to any degree of dilution of the blood, and is simple to re- 
member : one always counts a number of fields equal to the 
number of times the blood has been diluted, and adds two ciphers. 

It is sometimes impossible to obtain the proper size of 
field with the objectives and eye-pieces at hand. In such case 
place a cardboard disc with a circular opening upon the dia- 
phragm of the eye-piece, and adjust the size of the field by 
drawing out the tube. The circular opening can be cut with 
a cork-borer. 

Diluting Fluids. — The diluting fluid should dissolve the 
red corpuscles so that they will not obscure the leukocytes. 
The simplest fluid is a 0.5 per cent, solution of acetic acid. 
More satisfactory is the following: glacial acetic acid, i c.c; 
I per cent, aqueous solution of gentian-violet, i c.c. ; distilled 
water, 100 c.c. These solutions must be filtered frequently. 

VI. ENUMERATION OF BLOOD-PLAQUES 

The av^erage normal number of plaques is variously 
given as 200,000 to 700,000 per cubic millimeter of 
blood. Many of the counts were obtained by workers 
who used unreliable methods. Using their new method, 
Wright and Kinnicutt find the normal average to range 
from 263,000 to 336,000. Physiologic variations are 



214 THE BLOOD 

marked; thus, the number increases as one ascends to a 
higher altitude, and is higher in winter than in summer. 
There are unexplained variations from day to day; 
hence a single abnormal count should not be taken to 
indicate a pathologic condition. 

Pathologic variations are often very great. Owing to 
lack of knowledge as to the origin of the platelets and to 
the earlier imperfect methods of counting, the clinical 
significance of these variations is uncertain. The fol- 
lowing facts seem, however, to be established: 

(a) In acute infectious diseases the number is sub- 
normal or normal. If the fever ends by crisis, the crisis 
is accompanied by a rapid and striking increase. 

(b) In secondary anemia plaques are generally in- 
creased, although sometimes decreased In pernicious 
anemia they are always greatly diminished, and an 
increase should exclude the diagnosis of this disease. 

(c) They are decreased in chronic lymphatic leukemia, 
and greatly increased in the myelogenous form. 

(d) In purpura haemorrhagica the number is enor- 
mously diminished. 

Blood-plaques are difficult to count, owing to the 
rapidity with which they disintegrate and to their great 
tendency to adhere to any foreign body and to each 
other. 

Method of Kemp, Calhoun, and Harris. — Wash the 
finger well and allow a few minutes to elapse for the circu- 
lation to become normal. Prick the finger lightly with a 
blood-lancet, regulating the depth of the puncture so that 
the blood will not flow without gentle pressure. Quickly 
dip a clean glass rod into a vessel containing diluting and 
fixing fluid, and place two or three good-sized drops upon the 



ENUMERATION OF BLOOD-PLAQUES 215 

finger over the puncture. Then exert gentle pressure above 
the puncture so that a small drop of blood will exude into the 
fluid. Mix the two by passing the rod lightly several times 
over the surface of the blended drop. (Some workers first 
place a drop of the fluid upon the finger and then make the 
puncture through it, this necessitating less care as to depth of 
the puncture.) Now transfer a drop of the diluted blood from 
the finger to a watch-glass which contains two or three drops 
of the fluid, and mix well. From this, transfer a drop to the 
counting slide of the Thoma-Zeiss hemocytometer, and cover. 
An ordinary thin cover will answer for this purpose, and is 
preferable because it allows the use of a higher power object- 
ive. Allow the slide to stand for at least five minutes, and 
then with a 4 mm. or higher objective count the plaques 
and the red corpuscles in a definite number of squares. At 
least 100 plaques must be counted. The number of red cor- 
puscles per cubic millimeter of blood having been previously 
ascertained in the usual manner (p. 195), the number of 
plaques can easily be calculated by the following equation: 

r -.p -.-.R-.P ; andP = ^Jl_?. 

r represents the number of red corpuscles in any given 
number of squares; p, the number of plaques in the same 
squares; R, the total number of red corpuscles per c.mm. of 
blood; and P, the number of plaques per c.mm. 

Beginners are apt to take too much blood and not to dilute 
it enough. Best results are attained when there are only one 
or two plaques in a small square. With insufladent dilution, 
the platelets are more or less obscured by the red cells. 

The following diluting and fixing fluid is recommended: 

Formalin 10 c.c. 

I per cent, aqueous solution sodium chlorid 150 c.c. 

(Color with methyl-violet if desired.) 



2l6 THE BLOOD 

This fluid is cheap and easily prepared, and keeps indefi- 
nitely. It fixes the plaques quickly without clumping, and 
does not clump nor decolorize the reds. It has a low specific 
gravity, and hence allows the j^laques to settle upon the ruled 
area along with the reds. Fluids of high specific gravity 
cause the plaques to float so that they do not appear in the 
same plane with the reds and the ruled lines. 

Method of Wright and Kinnicutt. — This new method is 
simple, appears to be accurate, and certainly yields uniform 
results. 

The plaques are counted with the Thoma-Zeiss hemocy- 
tometer already described, using a dilution of i : loo. The 
diluting fluid consists of two parts of an aqueous solution of 
" brilliant cresyl blue " (i : 300) and three parts of an aqueous 
solution of potassium cyanid (i : 1400). These two solutions 
must be kept in separate bottles and mixed and filtered im- 
mediately before using. After the blood is placed in the 
counting-chamber it is allowed to stand for ten minutes or 
longer in order that the plaques may settle. The plaques 
appear as rounded, lilac-colored bodies; the reds are decolor- 
ized, appearing only as shadows. 

The leukocytes are stained and may be counted at the 
same time. 

VII. STUDY OF STAINED BLOOD 

A. Making and Staining Blood-films 

1 . Spreading the Film.— Thin, even films are essential 
to accurate and pleasant work. They more than com- 
pensate for the time spent in learning to make them. 
There are certain requisites for success with any method : 
(a) The slides and covers must be perfectly clean: 
thorough washing with soap and water and rubbing 
with alcohol will usually suffice; (b) the drop of blood 



STUDY OF STAINED BLOOD 217 

must not be too large ; (c) the work must be done quickly, 
before coagulation begins. 

The blood is obtained from the finger-tip or the lobe 
of the ear, as for a blood count; only a very small drop is 
required. 

Ehrlich's Two Cover-glass Method. — This method is very 
widely used, but considerable practice is required to get good 
results. Touch a cover-glass to the top of a small drop of 
blood, and place it, blood side down, upon another cover- 




Fig. 81. — Spreading the film: two cover-glass method. 

glass. If the drop be not too large, and the covers be per- 
fectly clean, the blood will spread out in a very thin layer. 
Just as it stops spreading, before it begins to coagulate, 
pull the covers quickly but firmly apart on a line parallel to 
their plane (Fig. 81). It is best to handle the covers with 
forceps, since the moisture of the fingers may produce arti- 
facts. 

Two-slide Method. — Take a small drop of blood upon a 
clean slide about 5 inch from the end. Place the end of a 
second slide against the surface of the first at an angle of 
45°, and push it up against the drop of blood, which will 
immediately run across the end, filling the angle between the 



2l8 



THE BLOOD 



two slides. Now draw the " spreader slide " back along the 
other. The blood will follow. The thickness of the smear 
can be regulated by changing the angle. 




Fig. 82. — Spreading the film: two-slide method. 

Cigarette-paper Method. — This gives better results in the 
hands of the inexperienced than any of the methods in general 




Fig. 83. — Spreading the film. Cigarette-paper method applied to cover-glasses. 

use, and may be used with either slides or covers. A very thin 
paper, such as the " Zig-zag " brand, is best. Ordinary 



STUDY OF STAINED BLOOD 219 

cigarette paper and thin tissue-paper will answer, but do not 
give nearly so good results. 

Cut the paper into strips about f inch wide, across the ribs. 
Pick up one of the strips by the gummed edge, and touch its 
opposite end to the drop of blood. Quickly place the end 
which has the blood against a slide or a large cover-glass held 
in a forceps. The blood will spread along the edge of the 
paper. Now draw the paper evenly across the slide or cover. 
A thin film of blood will be left behind (Fig. 83). 

The films may be allowed to dry in the air, or may be 
dried by gently heating high above a flame (where one 
can comfortably hold the hand). Such films will keep 
for years, but for some stains they must not be more 
than a few weeks old. They must be kept away from 
flies — a fly can work havoc with a film in a few minutes. 

2. Fixing the Film.— In general, films must be "fixed" 
before they are stained. Fixation may be accomplished 
by chemicals or by heat. Those stains which are dis- 
solved in methyl-alcohol combine fixation with the staining 
process. 

Chemic Fixation. — Soak the film five to fifteen minutes 
in pure methyl-alcohol, or one-half hour or longer in equal 
parts of absolute alcohol and ether. One minute in i per cent, 
formalin in alcohol is preferred by some. Chemic fixation may 
precede eosin-methylene-blue and other simple stains. 

Heat Fixation. — This may precede any of the methods 
which do not combine fixation with the staining process; it 
must be used with Ehrlich's triple stain. The best method is 
to place the film in an oven, raise the temperature to 150° C, 
and allow to cool slowly. Without an oven, the proper 
degree of fixation is difficult to attain. Kowarsky has de- 
vised a small plate of two layers of copper (Fig. 84), upon 



220 THE BLOOD 

which the films are placed together with a crystal of urea. 
The plate is heated over a flame until the urea melts, and is 
then set aside to cool. This is said to give the proper degree 
of fixation, but in the writer's experience the films have always 
been underheated. He obtains better results by use of tar- 
taric acid crystals (melting-point, i68°-i7o° C). The pla.te, 
upon which have been placed the cover-glasses, film side 
down, and a crystal of the acid, is heated over a low flame until 
the crystal has completely melted. It should be held suffi- 
ciently high above the flame that the heating will require five 
to se\en minutes. The covers are then removed. Freshly 
made films of normal blood should be allowed to remain upon 




Fig. 84. — Kowarsky's plate for fixing blood (Klopstock and Kowarsky). 

the plate for a minute or two after heating has ceased. 
Fresh films require more heat than old ones, and normal blood 
more than the blood of pernicious anemia and leukemia. 

Fixation by passing the film quickly through a flame about 
twenty times, as is often done in routine work, is not recom- 
mended for beginners. 

3. Staining the Film. — The anilin dyes, which are 
extensively used in blood work, are of two general classes: 
basic dyes, of which methylene-blue is the t^pe; and acid 
dyes, of which eosin is the type. Nuclei and certain 
other structures in the blood are stained by the basic 
dyes, and are hence called basophilic. Certain struc- 



STUDY OF STAINED BLOOD 221 

tures take up only add dyes, and are called acidophilic, 
oxyphilic, or eosinophilic. Certain other structures are 
stained only by combinations of the two, and are called 
neutrophilic. Recognition of these staining properties 
marked the beginning of modern hematology. 

(i) Eosin and Methylene-blue. — In many instances 
this stain will give all the information desired. It is 
especially useful in studying the red corpuscles. Nuclei, 
basophilic granules, and all blood parasites are blue; 
erythrocytes are red or pink; eosinophilic granules, bright 
red. Neutrophihc granules and blood-plaques are not 
stained. 

(i) Fix the film by heat or chemicals. 

(2) Stain about five minutes with 0.5 per cent, alcoholic 
solution of eosin, diluted one-half with water. 

(3) Rinse in water, and dry between filter-papers. 

(4) Stain one-half to one minute with saturated aqueous 
solution of methylene-blue. 

(5) Rinse well, dry, and mount. Films upon slides may 
be examined with an oil-immersion objective without a cover- 
glass. 

(2) Ehrlich's Triple Stain. — This has been the stand- 
ard blood-stain for many years, but is now little used. 
It is probably the best for neutrophihc granules. It is 
difficult to make, and should be purchased ready pre- 
pared from a reHable dealer. Nuclei are stained blue 
or greenish blue; erythrocytes, orange; neutrophilic 
granules, violet; and eosinophilic granules, copper red. 
Basophilic granules and blood-plaques are not stained. 

Success in staining depends largely upon proper fixa- 
tion. The film must be carefully fixed by heat: under- 



222 THE BLOOD 

heating causes the erythrocytes to stain red; overheat- 
ing, pale yellow. The staining fluid is applied for five 
to fifteen minutes, and the preparation is rinsed quickly 
in water, dried, and mounted. Subsequent application 
of Lofiler's methylene-blue for one-half to one second will 
bring out the basophilic granules and improve the 
nuclear staining, but there is considerable danger of 
overstaining. 

(3) Polychrome Methylene-blue Eosin Stains. — These 
stains, outgrowths of the original Romanowsky method, 
have largely displaced other blood-stains for clinical 
purposes. They stain differentially every normal and 
abnormal structure in the blood. Most of them are 
dissolved in methyl alcohol and combine the fbdng with 
the staining process. Numerous methods of preparing 
and applying these stains have been devised. Two 
only need be given here: Wright's stain and Harlow's 
stain : 

Wright's Stain. — This is one of the best and is the 
most widely used in this country. The practitioner 
will find it best to purchase the stain ready prepared. 
Most microscopic supply-houses carry it in stock. 
Wright's most recent directions for its preparation and 
use are as follows:' 

Preparation. — To a 0.5 per cent, aqueous solution of 
sodium bicarbonate add methylene-blue (B. X. or " medicin- 
ally pure ") in the proportion of i gm. of the dye to each 100 
c.c. of the solution. Heat the mixture in a steam sterilizer 
at 100° C. for one full hour, counting the time after the ster- 
ilizer has become thoroughly heated. The mixture is to be 
contained in a flask, or flasks, of such size and shape that it 
^Journal of the American Medical Association, Dec. 3, 1910. 



STUDY OF STAINED BLOOD 223 

forms a layer not more than 6 cm. deep. After heating, 
allow the mixture to cool, placing the flask in cold water, 
if desired, and then filter it to remove the precipitate which 
has formed in it. It should, when cold, have a deep purple- 
red color when viewed in a thin layer by transmitted yellowish 
artificial light. It does not show this color while it is warm. 

To each 100 c.c. of the filtered mixture add 500 c.c. of a 
0.1 per cent, aqueous solution of " yellowish water-soluble " 
eosin and mix thoroughly. Collect the abundant precipitate 
which immediately appears on a filter. When the precipitate 
is dry, dissolve it in methylic alcohol (Merck's " reagent ") 
in the proportion of o.i gm. to 60 c.c. of the alcohol. In 
order to facilitate solution, the precipitate is to be rubbed up 
with the alcohol in a porcelain dish or mortar with a spatula 
or pestle. This alcoholic solution of the precipitate is the 
staining fluid. 

Application. — i. Cover the film with a noted quantity of 
the staining fluid by means of a medicine-dropper. 

2. After one minute add to the staining fluid on the film 
the same quantity of distilled water by means of a medicine- 
dropper and allow the mixture to remain for two or three 
minutes, according to the intensity of the staining desired. 
A longer period of staining may produce a precipitate. 
Eosinophilic granules are best brought out by a short period 
of staining. 

The quantity of the diluted fluid on the preparation should 
not be so large that some of it runs off. 

3. Wash the preparation in water for thirty seconds or 
until the thinner portions of the film become yellow or pink 
in color. 

4. Dry and mount in balsam. 

The stain is more conveniently applied upon cover- 
glasses than upon slides. Films much more than a month 
old do not stain well. In some localities ordinary tap- 



224 iHE BLOOD 

water will answer both for diluting the stain and for 
washing the film; in others, distilled water must be used. 
Different lots of Wright's fluid vary, and a few prelimin- 
ary stains should be made with each lot to learn its 
peculiarities. 

When properly applied, Wright's stain gives the fol- 
lowing picture (Plate VI): erythrocytes, yellow or pink; 
nuclei, various shades of bluish purple; neutrophilic 
granules, reddish Hlac; eosinophilic granules, bright red; 
basophilic granules of leukocytes and degenerated red 
corpuscles, very dark bluish purple; blood-plaques, 
dark lilac; bacteria, blue. The cytoplasm of lymphocytes 
is generally robin's-egg blue; that of the large mononu- 
clears may have a faint bluish tinge. Malarial parasites 
stain characteristically: the cytoplasm, sky-blue; the 
chromatin, reddish purple. 

Harlow's Stain. — Probably the simplest modification of 
the Romanowsky stain, both in preparation and method 
of use, is that devised by W. P. Harlow of the University 
of Colorado. It differentiates granules particularly 
well, but is not so satisfactory for demonstrating slight 
grades of polychromatophilia, because it usually gives all 
the red cells a faint bluish tinge. 

Preparation. — The stain consists of two solutions used 
separately: 

No. I. Eosin, yellowish, water soluble (Griibler) . i gram 

Methyl alcohol (Merck's reagent) loo c.c. 

No. 2. Methylene-blue (" B. X." or Ehrlich's 

rectified) (Griibler) i gram 

Methyl alcohol (Merck's reagent) loo c.c. 

Application. — (i) Stain the film without previous fixation 
for one minute with the eosin solution. 



STUDY OF STAINED BLOOD 22$ 

(2) Shake off the excess, allowing a very little to remain, 
and at once cover with the methylene-blue solution for one 
or two minutes. 

(3) Rinse quickly in distilled water, dry, and mount. 

It is well known that pathologic bloods will sometimes 
not stain well with fluids which are satisfactory for 
normal bloods. Doctors Peebles and Harlow have shown 
that the various polychrome methylene-blue-eosin stains 
can be modified to suit any blood by adding a little 
alkali or acid. The alkali used is a weak solution of 
" potassium hydrate by alcohol " in methyl alcohol; the 
acid, glacial acetic in methyl alcohol. In the case of the 
Harlow stain it is added to the methylene-blue solution 
only. The alkali solution also serves to " correct " old 
fluids which, by reason of development of formic acid 
in the methyl alcohol, do not stain sufficiently with the 
blue. In general a stain is satisfactory when both nuclei 
and neutrophilic granules are clearly defined. 

B. Study of Stained Films 

Much can be learned from stained blood-films. They 
furnish the best means of studying the morphology of the 
blood and blood parasites, and, to the experienced, they 
give a fair idea of the amount of hemoglobin and the 
number of red and white corpuscles. An oil-immersion 
objective is required. 

1. Erythrocytes.— Normally, the red corpuscles are 
acidophilic. The colors which they take with different 
stains have been described. When not crowded to- 
gether, they appear as circular, homogeneous discs, of 
nearly uniform size, averaging 7.5 fi in diameter (Fig. 
104). The center of each is somewhat paler than the 

15 



226 THE BLOOD 

periphery. The degree of pallor furnishes a rough index 
to the amount of hemoglobin in the corpuscle. As 
hemoglobin is diminished, the central pale area becomes 
larger and paler, producing the so-called " pessary 
forms " in which only the periphery of the cell is apparent. 
These forms indicate a low color index and are most 
abundant in chlorosis. Red cells are apt to be crenated 
when the film has dried too slowly. 

Pathologically, red corpuscles vary in size and shape, 
staining properties, and structure. 

(i) Variations in Size and Shape (See Plate IX and 
Fig. 104). — The cells may be abnormally small (called 
microcytes, 5 ;(/ or less in diameter); abnormally large 
{macrocytes, 10 to 12 u); or extremely large (megalocytes, 
12 to 20 ^). 

Variation in shape is often very marked. Oval, pyri- 
form, caudate, saddle-shaped, and club-shaped corpus- 
cles are common (Fig. 85). They are called poikilocytes, 
and their presence is spoken of as poikilocytosis. 

Red corpuscles which vary from the normal in size and 
shape are present in most symptomatic anemias, and in 
the severer grades are often very numerous. Irregular- 
ities are particularly conspicuous in leukemia and pernic- 
ious anemia, where, in some instances, a normal erythro- 
cyte is the exception. In pernicious anemia there is a 
decided tendency to large size and oval forms, and mega- 
locytes are rarely found in any other condition. 

(2) Variations in Staining Properties (See Plate 
IX). — These include polychromatophilia, basophiHc 
degeneration, and malarial stippling. With exception 
of polychromatophilia they are probably degenerative 
changes. 



STUDY OF STAINED BLOOD 



227 



(a) Polychromatophilia. — Some of the corpuscles par- 
tially lose their normal affinity for acid stains, and take 
the basic stain to greater or less degree. Wright's stain 
gives such cells a faint bluish tinge when the condition is 
mild, and a rather deep blue when severe. Sometimes 
only part of a cell is affected. A few polychromatophilic 
corpuscles can be found in marked symptomatic anemias. 




Fig. 85. — Abnormal red corpuscles: A, Poikilocytosis; B, basophilic granular degenera- 
tion; C, malarial stippling, the cell also containinij a tertian parasite ( X 1000) (courtesy of 
Dr. W. P. Harlow). 



They occur most abundantly in malaria, leukemia, and 
pernicious anemia. 

Polychromatophilia has been variously interpreted. 
It is thought by many to be evidence of youth in a cell, 
and hence to indicate an attempt at blood regeneration. 
There are probably several forms referable to different 
causes. 

{h) Basophilic Granular Degeneration {Degeneration oj 
Grawitz). — This is characterized by the presence, within 



228 THE BLOOD 

the corpuscle, of basophilic granules which vary in size 
from scarcely visible points to granules as large as those 
of basophilic leukocytes (Fig. 85). The number present 
in a red cell commonly varies in inverse ratio to their 
size. They stain deep blue with Wright's stain; not at 
all with Ehrlich's triple stain. The cell containing them 
may stain normally in other respects, or it may exhibit 
polychromatophilia. 

Numerous cells showing this degeneration are com- 
monly found in chronic lead-poisoning, of which they 




Fig. 86. — Normoblasts from cases of secondary anemia and leukemia (X looo) (photo- 
graphs by the author). 



were at one time thought to be pathognomonic. Except 
in this disease, the degeneration indicates a serious blood 
condition. It occurs in well-marked cases of pernicious 
anemia and leukemia, and, much less commonly, in very 
severe symptomatic anemias. 

(c) Malarial Stippling. — This term has been applied 
to the finely granular appearance often seen in red cor- 
puscles, which harbor malarial parasites (Plates VI and 
VII and Fig. 85). It is commonly classed with the degen- 
eration just described, but is probably distinct. Not 
all stains will show it. With Wright's stain it can be 



STUDY OF STAINED BLOOD 229 

brought out by staining longer and washing less than 
for the ordinary blood-stain. The minute granules stain 
reddish purple. 

(3) Variations in Structure. — The most important is 
the presence of a nucleus (Plates VI and IX and Fig. 
86). Nucleated red corpuscles, or erytkroblasts , are 
classed according to their size: microblasts, 5 |M or less in 
diameter; normoblasts, 5 to 10 a; and megaloblasts, above 
10 u. Microblasts and normoblasts contain one, rarely 
two, small round, sharply defined, deeply staining nuclei, 




Fig. 87. — Megaloblasts from a case of pernicious anemia ( X 1000) (courtesy of Dr. 
W. P. Harlow). 

often located eccentrically. Occasionally the nucleus is 
irregular in shape, " clover-leaf " forms being not infre- 
quent. The megaloblast (Fig. 87) is probably a distinct 
cell, not merely a larger size of the normoblast. Its 
nucleus is large, stains rather palely, has a delicate 
chromatin network, and often shows evidences of degen- 
eration (karyorrhexis, etc.). In ordinary work, however, 
it is safer to base the distinction upon size than upon 
structure. Any nucleated red cell, but especially the 
megaloblast, may exhibit polychromatophilia. 

Normally, erythroblasts are present only in the blood 



230 THE BLOOD 

of the fetus and of very young infants. In the adult, 
their presence in the circulating blood denotes an excess- 
ive demand upon the blood-forming organs to regenerate 
lost or destroyed red corpuscles. In response to this 
demand, immature and imperfectly formed cells are 
thrown into the circulation. Their number, therefore, 
is an indication of the extent to which the bone-marrow 
reacts rather than of the severity of the disease. Nor- 
moblasts occur in severe symptomatic anemia, leukemia, 
and pernicious anemia. They are most abundant in 
myelogenous leukemia. While always present in per- 
nicious anemia, they are often difficult to find. Megalo- 
blasts are found in pernicious anemia, and with extreme 
rarity in any other condition. They here almost inva- 
riably exceed the normoblasts in number, which is one of 
the distinctive features of the disease. Microblasts have 
much the same significance as normoblasts, but are 
less common. 

Cabot's ring bodies are ring- or figure-of-8 shaped 
structures which have been observed in certain of the 
red cells in pernicious anemia, lead-poisoning, and lym- 
phatic leukemia. They stain red with Wright's stain. 
Their nature is unknown. 

2. The Leukocytes.~An estimation of the number or 
percentage of each variety of leukocyte in the blood is 
called a dijjerential count. It probably yields more 
helpful information than any other single procedure in 
blood examinations. 

The differential count is best made upon a film stained 
with Wright's, Harlow's, or Ehrlich's stain. Go care- 
fully over the film with an oil-immersion lens, using a 
mechanical stage if available. Classify each leukocyte 



STUDY OF STAINED BLOOD 23 1 

seen, and calculate what percentage each variety is of 
the whole number classified. For accuracy, 50x3 to 1000 
leukocytes must be classified; for approximate results, 
300 are sufficient. Track of the count may be kept by 
placing a mark for each leukocyte in its appropriate 
column, ruled upon paper. Some workers divide a slide- 
box into compartments with slides, one for each variety of 
leukocyte, and drop a coffee-bean into the appropriate 
compartment when a cell is classified. When a conve- 
nient number of coffee-beans is used (any multiple of 
100), the percentage calculation is extremely easy. 

The actual number of each variety in a cubic milU- 
meter of blood is easily calculated from these percentages, 
and the total leukocyte count. An increase in actual 
number is an absolute increase; an increase in percentage 
only, a relative increase. It is evident that an absolute 
increase of any variety may be accompanied by a relative 
decrease. 

A record is generally kept of the number of nucleated 
red cells seen during a differential count of leukocytes. 

The usual classification of leukocytes is based upon 
their size, their nuclei, and the staining properties of the 
granules which many of them contain. It is not alto- 
gether satisfactory, but is probably the best which our 
present knowledge permits. 

The writer has foimd the table (Fig. 88, p. 232) 
very helpful in impressing this classification upon the 
student. It makes no attempt to indicate histogenetic 
Telationships. The leukocytes of normal blood fall into 
two groups, each including three types. The cells in 
Group I contain single, round, oval or horseshoe-shaped 
nuclei, and have few or no granules in their cytoplasm. 



232 



THE BLOOD 



The stippling of the cytoplasm shown in the diagram 
represents the finely granular appearance of protoplasiri, 
not true granulation. The cells in Group II are polymor- 
phonuclear and contain granules which are distinguished 
by their size and staining reactions. In its structure the 
chief abnormal leukocyte, the myelocyte, combines the 

LEUKOCYTES 

NORMAL /^DNORM^L 



MONONUCLE/7R ~ 
NON - GR/^NUL/Jr? 

1-LYMPHOCVTE 20-30°/„ 
2-L/5RGE MONONUCLE^RI 

3 Tr?/1N5ITI0N^L J 

POLYMORPHONUCLEAR 
GR/7NUL/7R 




MyELOOTE 



1-NEUTROPHIUCf 



2- EOSINOPHILIC iM 



3-B-^SOPHILIC 



1- ISIEUTPOPHILIC 60-75% § 



2 - EOSINOPHILIC 



2-4yo5i 



.'.-is-A 



3- BASOPHILIC 0.3% BfflJ 

Fig. 88. — Outline of the classification of leukocytes. 

two groups, being mononuclear like Group I, and gran- 
ular like Group II. 

(i) Normal Varieties. — {a) Lymphocjrtes. — These are 
small mononuclear cells without granules (Plates VI and 
X). They are about the size of a red corpuscle or 
slightly larger (6-io (u), and consist of a single, sharply 



10 HOITAMAJ^XH 

I oriT .t?.i;li\oT >tn; l)iixi Igiildolfi^oril ,^ :f:u'il>ijn f^-jjiiludol 
Ifitne bnfi ^gisl . vl ,j ;9l'>eoqio^ b^ a a< .no 



'^ 3no ,b3ii;lfjui ^m 

ifiluganJ MJi io baeo.j 

inaiHSnoi ^t{ioiu^i ^B^hunpaom ^3^^Bl ,0j ; jhun'to 

i) ni e3g£t2 luol ,^i ;BnBlcrti nEJnal !o •«i. ■ -"AunBTg 

h 313W rfJnuol bnB bnonv. ■srf) :3ji?.Biiiq iBhslum ndJTJJ adJ lo abya 

■ no* bsi ,8i ;nBiJi3) ^Mitoh Ho ^bt b moil n9>!«t gbil? ^mfw ^Ht moil 

I .gnikjqiJa l/ihi.' ' nn-j 

'iin()iw armol ji ;ub 



istingui^l 
'nicture the 
"inbines the 



Explanation of Plate VI 

Stained with Wright's stain. Ail drawn to same scale. 
I, Norma! red corpuscle for comparison; 2, normoblasts, one with 
lohulatcd nucleus; 3, megaioblast and microblast. The megaloblast 
shows a considerable degree of polychromatophilia; 4, blood-plaques, 
one lying uy.on a red corpuscle; 5, lymphocytes, large and small; 6, 
large mononuclear leukocyte; 7, transitional leukocyte;- 8, polymor- 
phonuclear neutrophilic leukocytes; 9, eosinophilic leukocytes, one 
ruptured; 10, basophilic leukocyte; 11, neutrophilic myelocyte. The 
granules are sometimes less numerous and less distinct than here shown; 
12, eosinophilic mvelocvtes; 13, basophilic myelocyte; 14, ''irritation" 
or "stimulation" form, with small vacuoles; 15, degenerated leukocytes: 
two polymorphonuclear neutrophiles, one ruptured, one swojlen and 
vacuolated; and a "basket cell" composed of an irregular meshwork 
of nuclear material; 16, large mononuclear leukocyte containing f)igment- 
granules; from a case of tertian malaria; 17, four stages in the asexuial 
cycle of the tertian malarial parasite: the second and fourth were drawn 
from the same slide taken from a case of double tertian; 18, red corpuscle 
containing tertian parasite and showing malarial stippling; 19, estivo- 
autumnal malarial parasites: two small ring forms within the same 
red cell, and a crescent with remains of the red corpuscle in its concavity. 



and gran- 

"Dicse are 
\ I and 



PLATE VI 



-•^'■-' 3 



4 



• 4 a 







"3»r 







10 







12 





13 




14 





16 



15 



17 



r*^ 



18 



G- 



19 



STUDY OF STAINED BLOOD 



233 



defined, deeply staining nucleus, surrounded by a narrow 
rim of protoplasm. The nucleus is generally round, but 
is sometimes indented at one side. Wright's stain gives 
the nucleus a deep purple color and the cytoplasm a pale 
robin's-egg blue in typical cells. Larger forms of lymph- 
ocytes are frequently found, especially in the blood of 
children, and are difficult to distinguish from the large 
mononuclear leukocytes. It is possible that the larger 




Fig. 89. — Lymphocytt).sis, c.ise of pertussis ( X 1000) (courtesy of Dr. W. P. Harlow). 



forms are young lymphocytes, which become smaller 
as they grow older. 

Lymphocytes are formed in the lymphoid tissues, 
including that of the bone-marrow. They constitute, 
normally, 20 to 30 per cent, of all leukocytes, or about 
1000 to 3000 per c.mm. of blood. They are more abun- 
dant in the blood of children. 

The percentage of lymphocytes is usually moderately 
increased in those conditions which give leukopenia, 



234 'i'llE BLOOD 

especially typhoid fe\'er, chlorosis, pernicious anemia, 
and many debilitated conditions. A marked increase, 
accompanied by an increase in the total leukocyte count, 
is seen in pertussis (Fig. 89) and lymphatic leukemia. In 
the latter the lymphocytes sometimes exceed 98 per cent. 
E.xophthalmic goiter commonly gives a marked relative 
lymphoc}tosis, while simple goiter does not affect the 
l}mphocytes. 

(h) Large Mononuclear Leukocjrtes (Plate VI). — 
These cells are two or three times the diameter of the 
normal red corpuscle. Each contains a single round or 
oval nucleus, often located eccentrically. The zone of 
protoplasm surrounding the nucleus is relatively wide. 
With Wright's stain the nucleus is less deeply colored 
than that of the lymphocyte, while the cytoplasm is very 
pale blue or colorless, and sometimes contains a few red- 
dish granules. The size of the cell, the width of the zone 
of cytoplasm, and the depth of color of the nucleus are 
the points to be considered in distinguishing between 
large mononuclears and lymphocytes. When large 
forms of the lymphocyte are present, the distinction is 
often difficult or impossible. It is then advisable to 
count the two cells together as lymphocytes. Indeed, 
they are regarded by some hematologists as identical. 

Large mononuclear leukocytes probably originate in 
the bone-marrow or spleen. Some hold that they are 
developed from the endothelial cells of the blood-vessels. 
They constitute 2 to 4 per cent, of the total number of 
leukocytes: 100 to 400 per c.mm. of blood. An increase 
is unusual except in malaria, where it is quite constantly 
observed, and where many of the cells contain engulfed 
pigment. 



STUDY OF STAINED BLOOD 



235 



{c) Transitional Leukocytes (Plate VI). — These are 
essentially large mononuclears with deeply indented or 
horseshoe-shaped nuclei. A few fine neutrophilic gran- 
ules are sometimes present in their cytoplasm. 

They are probably formed from the large mononuclears, 
and occur in the blood in about the same numbers. The 
two cells are usually counted together, constituting 4 to 
8 per cent, of the leukocytes. 




Fig. 90.— Marked polymorphonuclear neutrophilic leukocytosis ; ,s loooj (courtesy of 
Dr. W. P. Harlow). 

{d) Polymorphonuclear Neutrophilic Leukocytes (Plate 
VI). — There is usually no difficulty in recognizing 
"these cells. Their average size is somewhat less than 
that of the large mononuclears. The nucleus stains 
rather deeply, and is extremely irregular, often assuming 
shapes comparable to letters of the alphabet, E, Z, S, etc. 



236 THE BLOOD 

(Fig. 90). Frequently there appear to be several separ- 
ate nuclei, hence the widely used name, "polynuclear leu- 
kocyte." Upon careful inspection, however, delicate 
nuclear bands connecting the parts can usually be seen. 
The cytoplasm is relatively abundant, and contains 
great numbers of very fine neutrophilic granules (Fig. 
93). With Wright's stain the nucleus is bluish purple, 
and the granules reddish lilac. 

Polymorphonuclear leukocytes are formed in the bone- 
marrow from neutrophilic myelocytes. They constitute 
60 to 75 per cent, of all the leukocytes: 3000 to 7500 per 
c.mm. of blood. Increase in their number practically 
always produces an increase in the total leukocyte count, 
and has already been discussed under Polymorphonu- 
clear Leukocytosis. The leukocytes of pus, pus-corpuscles, 
belong almost wholly to this variety. 

A comparison of the percentage of polymorphonuclear 
cells with the total leukocyte count yields more informa- 
tion than a consideration of either alone. In a general 
way the percentage represents the severity of the infec- 
tion, or, more correctly, the degree of toxic absorption; 
while the total count indicates the patient's power of 
resistance. With moderate infection and good resisting 
powers the leukocyte count and the percentage of poly- 
morphonuclears are increased proportionately. When 
the polymorphonuclear percentage is increased to a nota- 
bly greater extent than is the total number of leuko- 
cytes, no matter how low the count, either very poor 
resistance or a very severe infection may be inferred. 

Gibson has suggested the use of a chart to express 
this relationship graphically (Fig. 91). Its arrangement 
is purely arbitrary, but it may be found helpful in inter- 



STUDY OF STAINED BLOOD 



237 



preting counts. An ascending line from left to right 
indicates an unfavorable prognosis in proportion as the 













."^o non 




/ 




<=)-'^ 










P-.'i 000 




/ 




on 










7>.o,ono 




/ 




RA 










1.*; 000 




_ / 




flO 










10, 000 




/ 


— 


■7-5 




, 


/ 


z 




.•^ 000 






— 


7T> 









— 





Total leuko- 
cyte count. 



Percentage of 
polymorpho- 
nuclears. 



Fig. 91. — Gibson chart with blood-count in two cases of appendicitis: Dotted line rep- 
resenting a mild case with prompt recovery; the continuous line, a very virulent strepto- 
coccic case with poor resistance, fKjritonitis, and early death. 



line approaches the vertical. All fatal cases show a ris- 
ing line. A descending or horizontal line suggests a very 
favorable prognosis. 



238 THE BLOOD 

It is a matter of observation that in the absence of 
acute infectious disease or of inflammation directly in 
the blood-stream {e. g., phlebitis, sigmoid sinusitis, septic 
endocarditis), a polymorphonuclear percentage of 85 or 
over points very strongly to gangrene or pus-formation 
somewhere in the body. On the other hand, excepting 
in children, where the percentage is normally low, pus 
is uncommon with less than 80 per cent, of polymorpho- 
nuclears. 

Normally, the cytoplasm of leukocytes stains pale 
yellow with iodin. Under certain pathologic conditions 
the cytoplasm of many of the polymorphonuclears stains 
diffusely brown, or contains granules which stain reddish 
brown with iodin. This is called iodophilia. Extracellu- 
lar iodin-staining granules, which are present normally, 
are more numerous in iodophilia. 

This iodin reaction occurs in all purulent conditions 
except abscesses which are thoroughly walled off and 
purely tuberculous abscesses. It is of some value in 
diagnosis between serous effusions and purulent exudates, 
between catarrhal and suppurative processes in the ap- 
pendix and Fallopian tube, etc. Its importance, how- 
ever, as a diagnostic sign of suppuration has been much 
exaggerated, since it may occur in any general toxemia, 
such as pneumonia, influenza, malignant disease, and 
puerperal sepsis. 

To demonstrate iodophilia, place the air-dried films in 
a stoppered bottle containing a few crystals of iodin until 
they become yellow. Mount in syrup of levulose and 
examine with an immersion objective. 

Arneth classifies pohonorphonuclear leukocytes into 
five groups, according to the number of lobes which the 



STUDY OF STAINED BLOOD 239 

nucleus shows. The percentage of cells in each group 
is fairly constant in health, but shows considerable varia- 
tion in disease. 

(e) Eosinophilic Leukocjrtes, or " Eosinophiles " 
(Plate VI). — The structure of these cells is similar to that 
of the polymorphonuclear neutrophiles, with the striking 
difference that, instead of fine neutrophilic granules, their 
cytoplasm contains coarse, round or oval granules having 
a strong affinity for acid stains. They are easily recog- 
nized by the size and color of the granules, which stain 
bright red with Wright's stain (Fig. 93). Their cyto- 
plasm has generally a faint sky-blue tinge, and the nu- 
cleus stains somewhat less deeply than that of the 
polymorphonuclear neutrophile. 

Eosinophiles are formed in the bone-marrow from 
eosinophilic myelocytes. Their normal number varies 
from 50 to 400 per c.mm. of blood, or i to 4 per cent, of 
the leukocytes. An increase is called eosinophilia, and is 
better determined by the actual number than by the 
percentage. 

Slight eosinophilia is physiologic during menstruation. 
Marked eosinophilia is always pathologic. It occurs in 
a variety of conditions, the most important of which are : 
infection by animal parasites; bronchial asthma; myeloge- 
nous leukemia; scarlet fever, and many skin diseases. 

{a) Eosinophilia may be a symptom of infection by any 
of the worms. It is fairly constant in trichinosis, uncinaria- 
sis, filariasis, and echinococcus disease. In this country 
an unexplained marked eosinophilia warrants examina- 
tion of a portion of muscle for Trichinella spiralis (p. 363). 

{h) True bronchial asthma commonly gives a marked 
eosinophilia during and following the paroxysms. This 



240 THE BLOOD 

is helpful in excluding asthma of other origin. Eosino- 
philes also appear in the sputum in large numbers. 

(c) In myelogenous leukemia there is almost invariably 
an absolute increase of eosinophiles, although, owing to 
the great increase of other leukocytes, the percentage is 
usually diminished. Dwarf and giant forms are often 
numerous. 

id) Scarlet fever is frequently accompanied by eosino- 
philia, which may help to distinguish it from measles. 

i^:: ^ m_^^>^ \-\ ^^ --i 

Fig. 92. — Basophilic leukocytes. .\\. the right is also a normoblast undergoing mitosis 
( X 1000) (photographs by the author). 

(e) Eosinophilia has been observed in a large number 
of skin diseases, notably pemphigus, prurigo, psoriasis, 
and urticaria. It probably depends less upon the vari- 
ety of the disease than upon its extent. 

(/) Basophilic Leukocytes or *' Mast-cells " (Plate 
VI). — In general, these resemble polymorphonuclear neu- 
trophiles except that the nucleus is less irregular and that 
the granules are larger and have a strong affinity for 
basic stains. They are easily recognized (Figs. 92 and 93). 
With Wright's stain the granules are deep purple, while 



STUDY OF STAINED BLOOD 24 1 

the nucleus is pale blue and is often nearly or quite hid- 
den by the granules, so that its form is difficult to make 
out. These granules are not colored by Ehrlich's stain. 
The nature of mast-cells is undetermined. They 
probably originate in the bone-marrow. They are least 
numerous of the leukocytes in normal blood, rarely ex- 
ceeding 0.5 per cent., or 25 to 50 per c.mm. A notable 
increase is Umited almost exclusively to myelogenous 
leukemia, where they are sometimes very numerous. 




B . . 

Fig. 93. — Ruptured leukocytes, showing relative size of granules: A, neutrophilic; B, 
eosinophilic; C, basophilic (X looo) (photographs by the author). 

(2) Abnormal Varieties. — (a) Myelocytes (Plate VI and 
Fig. 94). — These are large mononuclear cells whose cyto- 
plasm is filled with granules. Typically, the nucleus occu- 
pies about one-half of the cell, and is round or oval. It is 
sometimes indented, with its convex side in contact with 
the periphery of the cell. It stains rather feebly. The 
average diameter of this cell (about 15.75 !^) is greater 
than that of any other leukocyte, but there is much varia- 
tion in size among individual cells. Myelocytes are 
named according to the character of their granules — 
neutrophilic, eosinophilic, and basophilic myelocytes. 
These granules are identical with the corresponding 

16 



242 THE BLOOD 

granules in the leukocytes just described. The occur- 
rence of two kinds of granules in the same cell is rare. 

Myelocytes are the bone-marrow cells from which the 
corresponding granular leukocytes are developed. Their 
presence in the blood in considerable numbers is diagnos- 
tic of myelogenous leukemia. The neutrophilic form is 
the least significant. A few of these may be present in 
very marked leukocytosis or any severe blood condition, 
as pernicious anemia. Eosinophilic myelocytes are found 




A B 

Fig. 94. — Myelocytes from blood of myelogenous leukemia: A, Neutrophilic; B, eosino- 
philic (X 1000) (photographs by the author). 

only in myelogenous leukemia, where they are often very 
numerous. The basophilic variety is less common, and 
is confined to long-standing, severe myelogenous leu- 
kemia. 

(b) Atypic Forms. — Leukocytes which do not fit in 
with the above classification are not infrequently met, 
especially in high-grade leukocytosis, pernicious anemia, 
and leukemia. The nature of most of them is not clear, 
and their number is usually so small that they may be 



STUDY OF STAINED BLOOD 243 

disregarded in making a differential count. Among them 
are: 

(a) Border-line forms between polymorphonuclear 
neutrophils and neutrophilic myelocytes. 

(b) Small neutrophilic cells with a single round, 
deeply staining nucleus; they probably result from di- 
vision of polymorphonuclear neutrophiles. 

(c) " Irritation forms " — large non-granular mono- 
nuclear cells, whose cytoplasm stains fairly deep purple 
with Wright's stain, and intense brown with Ehrlich's: 




Fig. 9S- — A clu^U;r of blood-plaques and two plaques lying upon a red cell and simu- 
lating malarial parasites ( X 1000) (photograph by the author). 

they appear in the blood under the same conditions as 
myelocytes. 

(d) Degenerated forms: vacuolated leukocytes, or 
merely palely or deeply staining homogeneous or retic- 
ulated masses of chromatin (the so-called " basket-cells," 
Plate VI). 

■ 3. Blood=plaques.— These are not colored by Ehrlich's 
stain nor by eosin and methylene-blue. With Wright's 
stain they appear as spheric or ovoid, reddish to violet, 
granular bodies, 2 to 4 ^ in diameter. When well stained 



244 THE BLOOD 

a delicate hyaline peripheral zone can be distinguished. 
In ordinary blood-smears they are usually clumped in 
masses. A single platelet lying upon a red corpuscle may 
easily be mistaken for a malarial parasite (Plate VI and 

Fig. 95)- 

Blood-platelets are being much studied at present, but, 
aside from the facts mentioned under their enumeration 
(p. 213), little of clinical value has been learned. They 
have been variously regarded as very young red corpus- 
cles (the " hematoblasts" of Hayem), as disintegration 
products of leukocytes, as remnants of extruded nuclei 
of erythrocytes, and as independent nucleated bodies. 
The most probable explanation of their origin seems to 
be that of J. H. Wright, who, from his recent studies, 
regards them as detached portions of the cytoplasm of 
certain giant-cells of the bone-marrow and spleen. 

VIII. BLOOD PARASITES 
A. Bacteria 

Bacteriologic study of the blood is useful in many 
conditions, but in general, the elaborate technic involved 
takes it out of the reach of the clinician. As applied to 
the diagnosis of t\T3hoid fever, however, the technic of 
blood-cultures has been so simplified that it can be car- 
ried through by any one who is competent to do the 
simplest cultural work. 

Typhoid bacilli can be detected in the blood in prac- 
tically every case of typhoid fever in the first week of the 
disease; in about 80 to 85 per cent, of cases in the second 
week; and in decreasing percentages in the later weeks. 
The blood-culture, therefore, offers the most certain means 



BLOOD PARASITES 245 

of early diagnosis. It is in a sense complementary to the 
Widal reaction, the former decreasing and the latter 
increasing in reliability as the disease progresses. The 
blood-culture gives best results before the Widal appears, 
as one Would expect from the fact that the Widal test 
depends upon the presence of antibodies which destroy, 
or, at least injure, the bacilli. The two methods to- 
gether will establish the diagnosis in practically every 
case at any stage. Bacilli disappear from the blood in 
convalescence and reappear in a relapse. 

Technic of Blood-Cultures in Typhoid Fever. — The blood 
may be obtained in one of two ways: 

(a) With a spring-lancet make a deep puncture in the 
edge (not the side) of the lobe of the ear, as for a blood-count. 
Allow the blood to drop directly into a short culture-tube 
containing the bile medium. By gentle milking, 20 to 40 
drops can usually be obtained. This simple method of ob- 
taining blood is especially applicable during the first week of 
the disease when bacilli are abundant. Contamination with 
skin cocci is possible, but does not usually interfere when the 
bile medium is used. 

(b) In the later weeks of the disease a larger quantity of 
blood is needed. Prepare the skin on the front of the elbow, 
as for a minor operation, or simply rub well with alcohol. 
Tie a bandage tightly aroimd the upper arm, have the patient 
open and close the fist a few times, and when the veins are 
sufiiciently distended insert a hypodermic needle attached 
to a syringe into any vein that is prominent. The needle 
should go through the skin about J inch from the vein with 
the bevel at its tip uppermost, and should enter the vein 
from the side in a direction opposite to the blood-current (Fig. 
96). Unless too small a needle is used, blood will begin to 
rise in the syringe as soon as the needle has entered the vein. 



246 



THE BLOOD 



Suction is not necessary. When sufficient blood is obtained, 
the bandage is first removed, the needle is withdrawn, and 
the blood is allowed to run into a tube of culture-medium. 
It is usually easy to secure 5 to 10 c.c. of blood. The proced- 
ure causes the patient surprisingly little inconvenience, sel- 
dom more than does an ordinary hyj^odermic injection. 
There is rarely any difficulty in entering the vein except in 
children, and in adults when the arm is fat and the veins are 
small. If desired, one of the veins about the ankle can be 
used. Instead of a syringe one can use a large glass tube 




Fig. q6. — Method of obtaining blood for a blood-culture. 



which has been drawn out at the ends and one end ground to 
fit a " slip-on " needle. Either a large hypodermic needle or 
a small antitoxin needle may be used. These little instru- 
ments (Fig. 96) can be made by any glass-blower at a 
cost of about fifty cents, and several of them can be kept 
on hand in test-tubes sterilized ready for use. 

As special culture-medium, ox-bile is generally used. It 
favors the growth of the typhoid bacillus and retards the 
growth of other organisms. A good formula is given on p. 405. 

As soon as convenient after the blood is added, place the 
tubes in the incubator. After about twelve hours examine 



BLOOD PARASITES 247 

for motile bacilli. If none are found, transfer a few drops 
to tubes of bouillon or solidified blood-serum and incubate for 
twelve hours longer. If motile, Gram-negative bacilli are 
foimd; they are almost certainly typhoid bacilli. Further 
study is not necessary in practice, although desirable from 
a scientific point of view. The only bacilli which might cause 
confusion are the paratyphoid and colon bacilli, which can 
be distinguished by gas production in glucose media, indol 
production, and their effect upon litmus milk. The agglutin- 
ation test for the identity of the bacillus is not available 
clinically, since freshly isolated bacilli do not agglutinate 
well. 

B. Animal Parasites 

Of the animal parasites which have been found in the 
blood, five are interesting clinically: the spirochaeta of 
relapsing fever; trypanosomes; malarial parasites; filarial 
embryos; and the embryos of Trichinella spiralis. 

1. Spirochaeta recurrentis is described on p. 330. 

2. Trypanosoma Qambiense. — Various trypanosomes 
are common in the blood of fishes, amphibians, birds, and 
mammals (Fig. 113). They live in the blood-plasma and 
do not attack the corpuscles. In some animals they are 
apparently harmless; in others they are an important 
cause of disease. They are discussed more fully on p. 333. 

The trypanosome of human blood, Trypanosoma gam- 
biense (Plate VII), is an actively motile, spindle-shaped 
organism, two or three times the diameter of a red cor- 
puscle in length, with an undulating membrane which 
terminates at the anterior end in a long ilagellum. It can 
be seen with medium power objectives in fresh blood, but 
is best studied with an oil-immersion lens in preparations 
stained as for the malarial parasite. Human trypano- 



248 THE BLOOD 

somiasis is common in Africa. As a rule, it is a very 
chronic disease. " Sleeping sickness " is a late stage when 
the organisms have invaded the cerebrospinal fluid. 
Infection is carried by the tsetse fly, Glossina palpalis. 

3. The Malarial Parasites.— These protozoa belong to 
the Sporozoa (p. 338), order Hemosporidia, the mem- 
bers of which are parasites in the blood of a great 
variety of vertebrates. Three species, constituting the 
genus Plasmodium, are associated with malarial fever in 
man : Plasmodium vivax, P. malarice, and P. falciparum, 
the parasites respectively of the tertian, quartan, and 
estivo-autumnal types of malaria. The life histories of 
the three are so similar that they may well be described 
together. 

(i) Life Histories. — There are two cycles of develop- 
ment: one, the asexual, in the blood of man; and the 
other, the sexual, in the intestinal tract of a particular 
genus of mosquito, AnopJieles. 

(a) Asexual Cycle. — The young organism enters the 
blood through the bite of the mosquito. It makes its way 
into a red corpuscle, where it appears as a small, pale 
" hyaline " body. This body exhibits ameboid movement 
and increases in size. Soon, dark-brown granules derived 
from the hemoglobin of the corpuscle make their appear- 
ance within it. When it has reached its full size — filling 
and distending the corpuscle in the case of the tertian 
parasite, smaller in the others — the pigment granules 
gather at the center or at one side; the organism divides 
into a number of small hyaline bodies, the spores or 
merozoites; and the red corpuscle bursts, setting spores 
and pigment free in the blood-plasma. This is called 
segmentation. It coincides with, and by liberation of 



PLATE VII 




Trypanosoma gambiense (slide presented by Professor F. G. Novy). 




E 


1 

V 



Tertian malarial parasites, one red Estivo-autumnal malarial para- 

cell showing malarial stippling. sites, small ring forms and 

crescents. 




Spinu h.cta novyi. 
Animal parasites of the blood; X looo (photographs by the author). 



BLOOD PARASITES 249 

toxins causes, the paroxysm of the disease. A consider- 
able number of the spores are destroyed by leukocytes or 
other agencies; the remainder enter other corpuscles 
and repeat the cycle. Many of the pigment granules 
are taken up by leukocytes' In estivo-autumnal fever 
segmentation occurs in the internal organs and the seg- 
menting and larger pigmented forms are not seen in the 
peripheral blood. 

The asexual cycle of the tertian organism occupies 
forty-eight hours;. of the quartan, seventy- two hours; of 
the estivo-autumnal, an indefinite time — usually twenty- 
four to forty-eight hours. 

The parasites are thus present in the blood in great 
groups, all the individuals of which reach maturity and 
segment at approximately the same time. This explains 
the regular recurrence of the paroxysms at intervals cor- 
responding to the time occupied by the asexual cycle of 
the parasite. Not infrequently there is multiple infection, 
one group reaching maturity while the others are still 
young; but the presence of two groups which segment 
upon the same day is extremely rare. Fevers of longer 
intervals — six, eight, ten days — are probably due to the 
ability of the body, sometimes of itself, sometimes by aid 
of quinin, to resist the parasites, so that numbers suffi- 
cient to cause a paroxysm do not accumulate in the blood 
until after several repetitions of the asexual cycle. In 
estivo-autumnal fever the regular grouping, while usually 
present at first, is soon lost, thus causing "irregular 
malaria." 

(b) Sexual Cycle. — Besides the ameboid individuals 
which pass through the asexual cycle, there are present 
with them in the blood many individuals with sexual 



250 THE BLOOD 

properties. These are called gametes. They do not 
undergo segmentation, but grow to adult size and remain 
inactive in the blood until taken up by a mosquito. 
Many of them are apparently extracellular, but stained 
preparations usually show them to be surrounded by the 
remains of a corpuscle. In tertian and quartan malaria 
they cannot easily be distinguished from the asex- 
ual individuals until a variable time after the blood 
leaves the body, when the male gamete sends out 
one or more flagella. In estivo-autumnal malaria the 
gametes take distinctive ovoid and crescentic forms, and 
are not difficult to recognize. They are very resistant to 
quinin and often persist in the blood long after the 
ameboid forms have been destroyed, but are probably 
incapable of continuing the disease until they have passed 
through the cycle in the mosquito. 

When a malarious person is bitten by a mosquito, the 
gametes are taken with the blood into its stomach. Here 
a flagellum from the male unites with the female, which 
soon thereafter becomes encysted in the wall of the intes- 
tine. After a time it ruptures, liberating many minute 
rods, or sporozoites, which have formed within it. These 
migrate to the salivary glands, and are carried into the 
blood of the person whom the mosquito bites. Here they 
enter red corpuscles as young malarial parasites, and the 
majority pass through the asexual cycle just described. 

The sexual cycle can take place only within the body 
of one genus of mosquito. Anopheles. Absence of this 
mosquito from certain districts explains the absence of 
malaria. It is distinguished from our common house- 
mosquito, Culex, by the relative lengths of proboscis and 
palpi (Fig. 97), which can be seen \\ith a hand-lens, by 



BLOOD PARASITES '-^^flAf-.y ^SJ 

r 

its attitude when resting, and by its dappled wing (Pi^.f /: j 
98). Anopheles is strictly nocturnal in its habits; it 
usually flies low, and rarely travels more than a few 
hundred yards from its breeding-place, although it may 
be carried by winds. These facts explain certain peculi- 
arities in malarial infection; thus, infection occurs prac- 
tically only at night; it is most common near stagnant 
water, especially upon the side toward which the pre- 
vailing winds blow; and the danger is greater when per- 




Fig. 97. — Mosquitoes — Culex (i) and Anopheles (2) (Bergey). 

sons sleep upon or near the ground than in upper stories 
of buildings. The insects frequently hibernate in warmed 
houses, and may bite during the winter. A mosquito 
becomes dangerous in eight to fourteen days after it 
bites a malarious person, and remains so throughout 
its Hfe. 

(2) Detection. — Search for the malarial parasite may 
be made in either fresh blood or stained films. If possible, 
the blood should be obtained a few hours before the chiU 
— never during it nor within a few hours afterward, since 



252 



r<\ 



THE BLOOD 



at that time (in single infections) only the very young, 
unpigmented forms are present, and these are the most 
difficult to find and recognize. Sometimes many para- 
sites are found in a microscopic field; sometimes, especi- 






Fig. 98. — Showing, on the left. Anopheles in resting position, its dappled wing, and 
the position of its larvae in water; on the right, Culex in resting position, its plain wing, 
and the position of its larvae in water. The arrows indicate the directions taken by the 
larvae when the water is disturbed (Abbott). 



ally in estivo-autumnal infection, owing to accumulation 
in internal organs, careful search is required to find any, 
despite very severe symptoms. Quinin causes them 
rapidly to disappear from the peripheral blood, and few 
or none may be found after its administration. In the 



BLOOD PARASITES, ~ '"'^/^/^/ ^ 

absence of organisms, the presence of pigment granul6&f A f 
within leukocytes — polymorphonuclears and large mono- ^. . 
nuclears — may be taken as presumptive evidence of 
malaria. Pigmented leukocytes (Plate VI) are most 
numerous after a paroxysm. 

(a) In Fresh Unstained Blood (Plate VIII) . — Obtain a 
small drop of blood from the finger or lobe of the ear. 
Touch the center of a cover-glass to the top of the drop 
and quickly place it, blood side down, upon a slide. If 
the slide and cover be perfectly clean and the drop not 
too large, the blood will spread out so as to present only 
one layer of corpuscles. Search with an oil-immersion 
objective, using very subdued light. 

The young organisms appear as small, round, ring-like 
or irregular, colorless bodies within red corpuscles. . The 
light spots caused by crenation and other changes in the 
corpuscles are frequently mistaken for them, but are 
generally more refractive or have more sharply defined 
edges. The older forms are larger colorless bodies con- 
taining granules of brown pigment. In the case of the 
tertian parasite, these granules have active vibratory 
motion, which renders them conspicuous; and as the 
parasite itself is very pale, one may see only a large pale 
corpuscle in which fine pigment granules are dancing. 
Segmenting organisms, when typic, appear as rosets, 
often compared to daisies, the petals of which represent 
the segments, while the central brown portion represents 
the pigment. Tertian segmenting forms are less fre- 
quently typic than quartan. Flagellated forms are not 
seen until ten to twenty minutes after the blood has left 
the vessels. As Cabot suggests, one should, while search- 
ing, keep a sharp lookout for unusually large or pale cor- 



254 THE BLOOD 

pu.sdes, and for anything which is brown or black or in 
motion. 

(b) In Stained Films (Plates VI and VII). — Recogni- 
tion of the parasite, especially the young forms, is much 
easier in films stained by Wright's or some similar stain 
than in fresh blood. When very scarce, they may some- 
times be found, although their structure is not well shown 
b\- the method of Ruge. This consists in spreading a very 
thick layer of blood, drying, placing for a few minutes in 
a fluid containing 5 per cent, formalin and i per cent, 
acetic acid, which removes the hemoglobin and fixes the 
smear, rinsing, drying, and finally staining. Carbol- 
thionin is very useful for this purpose. If Wright's 
stain be used in this method, it is recommended that the 
preparation be subsequently stained for a half-minute 
with borax-methylene-blue (borax, 5 ; methylene-blue, 2 ; 
water, 100). 

In films which are properly stained with W^right's fluid 
the young organisms are small, round, ring-like or irreg- 
ular, sky-blue bodies, each with a very small, sharply de- 
fined, reddish-purple chromatin mass. Many structures 
— deposits of stain, dirt, blood-plaques lying upon red 
cells (Fig. 95), etc.- -may simulate them, but should not de- 
ceive one who looks carefully for both the blue cytoplasm 
and the reddish-purple chromatin. A plaque upon a red 
corpuscle is surrounded by a colorless zone rather than by 
a distinct blue body. Young estivo-autumnal parasites 
commonly take a " ring " form (the chromatin mass rep- 
resenting the jewel), which is infrequently assumed by 
the other varieties. The older tertian and quartan or- 
ganisms show larger sky-blue bodies with more reticular 
chromatin, and contain brown granules of pigment, which, 



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Explanation of Plate VIII 

Various forms of malarial parasites (unstained) (Thayer and Hewetson). 
I to lo, inclusive, Tertian organisms; ii to 17, inclusive, quartan 
organisms; 18 to 27, inclusive, estivo-autumnal organisms; i, young 
hyaline form; 2, hyaline form with beginning pigmentation; 3, pig- 
mented form; 4, full-grown pigmented form; 5, 6, 7, 8, segmenting 
forms; g, extracellular pigmented form; 10, flagellate form; 11, young 
hyaline form; 12, 13, pigmented forms; 14, fully developed pigmented 
form; 15, 16, segmenting forms; 17, flagellate form; 18, 19, 20, ring-like 
and cross-like hyaline forms; 21, 22, pigmented forms; 23, 24, segmenting 
forms; 25, 26, 27, crescents. 



PLATE VIII 






3 4 5 6 






// /? /? M 



r 



15 J6 17 



^i::^ 



^J< 



JS 19 20 2 J 22 



^i 24 25 26 27 



BLOOD PARASITES 255 

however, is less evident than in the living parasite. The 
chromatin is often scattered through the cytoplasm or 
apparently outside of it, and is sometimes difficult to see 
clearly. Typical '' segmenters " present a ring of rounded 
segments or spores, each with a small, dot-like chromatin 
mass. With the tertian parasite, the segments more fre- 
quently form an irregular cluster. The pigment is col- 
lected near the center or scattered among the segments. 
In estivo-autumnal fever usually only the small " ring 
bodies " and the crescentic and ovoid gametes are seen 



^ 




Fig. gg. — Filarial embryos in blood. Stained. Red corpuscles decolorized; a few leuko- 
cytes remain (X 200) (photographs by the author). 

in the blood. The gametes are easily recognized. Their 
length is somewhat greater than the diameter of a red 
corpuscle. Their chromatin is usually centrally placed, 
and they contain more or less coarse pigment. The re- 
mains of the red cell often form a narrow rim around them 
or fill the concavity of the crescent. 
- While the parasites are more easily found in stained 
preparations, the varieties are more easily differentiated 
in fresh blood. The chief distinguishing points are 
included in the table on page 256. 



256 THE BLOOD 

VARIETIES OF THE MALARIAL ORGANISM 



Tertian. 


Quartan. 


EsTivo-AtrnniNAL, 


Asexual cycle, forty-eight 
hours. 


Seventy-two hours. 


Usually twenty-four to 
forty-eight hours. 


Substance pale, trans- 
parent, comparable to 
hyaline tube-cast. 

Outline indistinct. 

Ameboid motion ac- 
tive. 

Mature asexual form 
large; fills and often dis- 
tends corpuscle. 

Pigment -granu'^s 
fine, brown, scattered 
throughout. Very ac- 
tive dancing motion. 

Segmenting body 
rarely assumes typical 
"daisy" form. 15 to 
20 segments. 

Gametes resemble 
asexual forms. 

Red corpuscles pale 
and swollen. 


Highly refractive, 
comparable to waxy 
tube-cast. 

Distinct. 

Sluggish. 

Smaller, 

Much coarser, darker 
in color, peripherally ar- 
ranged. Motion slight. 

Usually typical 
"daisy." 6 to 12 seg- 
ments. 

Same as tertian. 

Generally darker than 
normal. 


Highly refractive. 

Distinct. 
Active. 

Young forms, only, 
in peripheral blood. 

Very few, minute, 
inactive. Distinctly 
pigmented forms sel- 
dom seen. 

Not seen in peri- 
pheral blood. 

Appear in blood as 
distinctive ovoids and 
crescents. 

Dark, often bronzed. 



4. Filarial Embryos.— A description of the filariae 
whose embryos appear in the blood will be found on 
P- 356. 

The embryos can be seen in stained preparations, (Fig. 
99), but are best found in fresh unstained blood. A 
rather large drop is taken upon a slide, covered, and ex- 
amined with a low power. The embryo can be located 
by the commotion which its active motion produces 



SERUM REACTIONS 257 

among the corpuscles. This motion consists almost 
wholly in apparently purposeless lashing and coiling 
movements, and continues for many hours. 

5. Embryos of Trichinella Spiralis.— The worm and 
its life-history are described on page 363. It has re- 
cently been shown that diagnosis of trichiniasis can fre- 
quently be made by detection of the embryos in the 
blood during their migration to the muscles. Of eleven 
such examinations which have been reported within the 
past two and a half years, six were positive. The earliest 
time at which the embryos were found was the sixth day 
after the onset of symptoms; the latest, the twenty- 
second day. 

The method is very simple. One to 10 c.c. of blood 
are obtained from the ear or a vein, as described on page 
245, and mixed with ten times its volume of 3 per cent, 
acetic acid. The mixture is centrifugalized, and large 
drops of the sediment are placed on sUdes, covered, and 
searched with a low-power objective. The embryos are 
not difficult to recognize. They are about 1 25 |tz long and 
6/z broad. 

IX. SERUM REACTIONS 

*I. Agglutination.— In the blood-serum of persons 
suffering from certain infectious diseases there exist sol- 
uble bodies, called agglutinins, which have the property of 
rendering non-motile and clumping the specific micro- 
organism of the disease, and have little or no influence 
upon other bacteria. This " agglutination " takes place 
even when the blood is greatly diluted. Undiluted nor- 
mal blood can agglutinate most bacteria, but loses this 
power when diluted to any considerable degree. These 

17 



258 



THE BLOOD 



facts are taken advantage of in the diagnosis of several 
diseases. 

When appKed to the diagnosis of typhoid fever, the 
phenomenon is known as the Widal reaction. As yet, it 
is the only agglutination reaction which has any practical 
value for the practitioner. 

Either blood-serum or the whole blood may be used. 
Serum is the better. To obtain it, it is convenient to use 
little vials, such as can be made by breaking off the lower 




Fig. loo. — Method of obtaining blood in a Wright capsule: A, Filling the capsule; B, 
the bulb has been warmed and the capillary end sealed in a flame; C, cot)ling of the capsule 
has drawn the blood to the sealed end; D, the serum has separated, and the top of the cap- 
sule has been broken off. 



half -inch of the tubes which have contained pepton- 
izing powder. They must, of course, be well cleaned. 
One of these is filled to a depth of about \ inch from 
a puncture in the finger or the ear. and is set aside for a 
few hours. When the clot has separated, it is picked out 
with a needle, leaving the serum. It is, however, more 
satisfactory to obtain the blood in a Wright capsule 
(Fig. loo). This capsule is easily made from a piece of 
glass- tubing as indicated in Fig. i6i. 



SERUM REACTIONS 259 

One drop of the serum is then added to nine drops 
of normal salt solution, making a dilution of i : 10. 
Distilled water may be used for dilution, but is more 
liable to cause error. The dilution can be more accu- 
rately made in the leukocyte pipet of the Thoma- 
Zeiss instrument. When the whole blood is used, it 
can be secured in this pipet and at once diluted with 
the salt solution. When it must be transported a con- 
siderable distance, dried blood is most convenient. A 
large drop is allowed to dry upon a clean slide or unglazed 
paper. It will keep for months without losing its ag- 
glutinating power. When ready to make the test, the 
dried stain is dissolved in ten drops of normal salt solu- 
tion, care being taken that the drops are about the same 
size as the original drop of blood. 

The reaction can be detected either microscopically or 
macroscopically : 

Microscopic Method. — (i) The blood or serum having 
been obtained and diluted i : 10 as just described, mix it with 
a bouillon culture of the typhoid bacillus to any desired 
dilution. One drop of each makes a blood-dilution of i : 20, 
etc. The culture should be between eighteen and twenty- 
four hours old, and the bacilli must be actively motile. A 
stock agar culture should be kept at room temperature, and 
bouillon tubes inoculated the day before the examination is to 
be made. Agar cultures can be purchased from dealers in 
biologic products. They must be renewed monthly. 

Instead of the bouillon culture, McFarland recommends 
the use of a suspension made by removing some of the growth 
from the surface of a fresh agar culture and mixing it well 
with a little sterile water. It is then necessary to examine the 
suspension microscopically to make sure that there are no 
natural clumps. 



26o THE BLOOD 

(2) Place a few drops of the mixture of blood and culture 
upon a perfectly clean slide and apply a cover-glass. The 
cover may be ringed with vaselin to prevent evaporation, but 
this is not usually necessary. 

(3) Examine at intervals with a high dry lens — a 4 mm. 
will answer very well. The light must be very subdued. 
At first the bacilli should be actively moving about. If the 
blood be from a case of typhoid, they will gradually lose their 
motion and gather together in clumps (Fig. loi). The clumps 
should be large, and the few bacilli remaining isolated should 






Fig. 101. — Showing clumping of typhoid bacilli in the Widal reaction. At one point a 
crenated red blood-corpuscle is seen (Wright and Brown). 

be motionless. Pseudoreactions, in which there are a few 
small clumps of bacilli whose motion is not entirely lost, 
together with many freely moving bacilli scattered through- 
out the field, should not mislead. As a control, a drop 
of the culture should always be examined before making 
the test. 

Normal blood may produce clumping if time enough be 
allowed. The diagnostic value of a positive reaction is, there- 
fore, impaired unless clumping takes place within a limited 
time. With dilution of i : 40 the time limit should not exceed 



SERUM REACTIONS 26 1 

forty-five minutes; with i : 80, one and one-half hours. 
Tests based upon lower dilution than i : 40 are probably not 
reliable. 

Macroscopic Method. — The principle is the same as that 
of the microscopic method. Clumping of the bacilli causes 
a flocculent precipitate, which can be seen with the naked 
eye. A dead culture gives the same results as a living one. 
This method is as reliable as the microscopic and is more 
convenient for the practitioner, although it requires more 
time. 

Dead cultures, together with apparatus for diluting the 
blood, are put up at slight cost by various firms, under the 
names of typhoid diagnosticum, typhoid agglutometer, etc. 
Full directions accompany these outfits and need not be 
repeated here. 

Recently, Bass and Watkins have described a modification 
of the macroscopic method (using very concentrated sus- 
pensions of the bacilli) by which the test can be applied at 
the bedside. Clumping occurs within two minutes, ' The 
apparatus has been put upon the market by Parke, 
Davis & Co. 

The Widal reaction is positive in over 95 per cent, of all 
cases of typhoid fever. It may, rarely, be positive in 
other conditions, owing, sometimes at least, to faulty 
technic. It seldom appears before the fifth or sixth day; 
usually during the second week, but sometimes not until 
convalescence. It is, therefore, of less value in early 
diagnosis than is the blood-culture (p. 244). When it 
once appears it remains during the whole course of the 
disease, and frequently persists for years. 

2. Opsonins. — That phagocytosis plays an important 
part in the body's resistance to bacterial invasion has 
long been recognized. According to Metchnikoff, this 



262 THE BLOOD 

property of leukocytes resides entirely within themselves, 
depending upon their own vital activity. The studies 
of Wright and Douglas, upon the contrary, indicate 
that the leukocytes are impotent in themselves, and can 
ingest bacteria only in the presence of certain substances 
which exist in the blood-plasma. These substances have 
been named opsonins. Their nature is undetermined. 
They probably act by uniting with the bacteria, thus 
preparing them for ingestion by the leukocytes ; but they 
do not cause death of the bacteria, nor produce any 
appreciable morphologic change. They appear to be 
more or less specific, a separate opsonin being necessary 
for phagocytosis of each species of bacteria. There are, 
moreover, opsonins for other formed elements — red 
blood-corpuscles, for example. It has been shown that 
the quantity of opsonins in the blood can be greatly 
increased by inoculation with dead bacteria. 

To measure the amount of any particular opsonin in the 
blood Wright has devised a method which involves many 
ingenious and delicate technical procedures. Much skill, 
such as is attained only after considerable training in lab- 
oratory technic, is requisite, and there are many sources 
of error. It is, therefore, beyond the province of this 
work to recount the method in detail. In a general way 
it consists in: {a) Preparing a mixture of equal parts of 
the patient's blood-serum, an emulsion of the specific 
micro-organism, and a suspension of washed leukocytes; 
{h) preparing a similar mixture, using serum of a normal 
person; {c) incubating both mixtures for a definite length 
of time; and id) making smears from each, staining, and 
examining with an oil-immersion objective. The num- 
ber of bacteria which have been taken up by a definite 



SERUM REACTIONS 263 

number of leukocytes is counted, and the average number 
of bacteria per leukocyte is calculated; this gives the 
"phagocytic index." The phagocytic index of the blood 
under investigation, divided by that of the normal 
blood, gives the opsonic index of the former, the opsonic 
index of the normal blood being taken as i. Simon re- 
gards the percentage of leukocytes which have ingested 
bacteria as a more accurate measurement of the amount 
of opsonins than the number of bacteria ingested, be- 
cause the bacteria are apt to adhere and be taken in in 
clumps. 

Because of its simplicity the clinical laboratory 
worker will prefer some modification of the Leishman 
method, which uses the patient's own leukocytes. It 
is, perhaps, as accurate as the original method of Wright, 
although variations in the leukocyte count have been 
shown to affect the result. Two pipets like those 
shown in Fig. 164 are used. 

(i) Make a suspension of the specific organism by mixing 
a loopful of a young agar culture with i c.c. of a solution con- 
taining I per cent, sodium citrate and 0.85 per cent, sodium 
chlorid. Thoroughly break up all clumps by sucking the 
fluid in and forcing it out of one of the capillary pipets held 
vertically against the bottom of the watch-glass. 

(2) Puncture the patient's ear, wipe off the first drop of 
blood, and from the second draw blood into the other pipet 
to the grease pencil mark, let in a bubble of air, and draw in 
-the same amount of bacterial suspension. 

(3) Mix upon a slide by drawing in and forcing out of the 
pipet. 

(4) Draw the mixture high up in the pipet, seal the tip 
in the flame, and place in the incubator for fifteen minutes. 



264 THE BLOOD 

(5) Repeat steps 2, 3, and 4 with the blood of a normal 
person. 

(6) After incubation, break off the tip of the pipet, mix the 
blood-bacteria mixture, and spread films on slides. 

(7) Stain with Wright's or Harlow's blood-stain. 

(8) With an oil-immersion lens count the bacteria which 
have been taken in by 100 leukocytes, and calculate the aver- 
age number per leukocyte. Divide the average for the 
patient by the average for the normal person. This gives 
the opsonic index. If in the patient's blood there was an 
average of 4 bacteria per leukocyte, and in the normal blood 
5 bacteria per leukocyte, the opsonic index would be | or 0.8. 

Wright and his followers regarded the opsonic index 
as an index of the power of the body to combat bacterial 
invasion. They claimed very great practical importance 
for it as an aid to diagnosis and as a guide to treatment 
by the vaccine method. This method of treatment con- 
sists in increasing the amount of protective substances 
in the blood by injections of normal salt suspensions of 
dead bacteria of the same species as that which has 
caused and is maintaining the morbid process, these 
bacterial suspensions being called 'Vaccines." Vaccine 
Therapy (Chapter IX) has taken a permanent place 
among our methods of treatment of bacterial infections, 
particularly of those which are strictly local, but the 
opsonic index is now little used either as a measure of 
resisting power or as an aid to diagnosis and guide to 
treatment. 

3. Wassermann Reaction.'— The Wassermann test 
for syphilis, like the Widal test for typhoid fever, de- 

* By Clough T. Burnett, Professor of Bacteriology, University of Col- 
oradg. 



SERUM REACTIONS 265 

pends upon the detection in the patient's blood-serum of 
specific antibodies, agglutinins in the case of typhoid, 
immune bodies or amboceptors in the case of syphilis. 
These antibodies have been produced by the tissues in 
response to the entrance of the invading organism. If 
they are present, it is assumed that the patient has or 
has had syphiHs. The Wassermann test is, however, 
much more complicated than the Widal test, and can be 
properly performed only by a trained laboratory worker. 
It is the aim here to explain only the general principles 
of the method, together with its clinical significance. 
For a proper understanding of the test the principles 
of bacteriolysis and hemolysis must first be presented. 

Bacteriolysis and Hemolysis. — In 1894 Pfeiffer, work- 
ing with guinea-pigs immunized to cholera, found that 
when living cholera germs were introduced into the 
peritoneal cavity of an immune animal they lost their 
motility within a few minutes, and very shortly were 
seen to disintegrate and go into complete solution. 
This has been known as Pfeiffer's phenomenon, or 
bacteriolysis. It was later demonstrated that this reac- 
tion could take place outside the animal body if the 
bacteria were mixed in the test-tube with the blood- 
serum or peritoneal fluid of a cholera immune animal. 
Subsequent researches showed that while an old or 
heated immune serum failed to cause this solution of 
the bacteria, upon the addition of a normal fresh serum 
this property returned. This addition of a normal 
serum to a serum which has lost its solvent action is 
called reactivation of the serum. These changes may 
best be demonstrated by the following chart; 



266 



3 




THE BLOOD 






Bacteriolysis. 


Immune serum, 


fresh 


+ bacteria = solution. 


Normal " 


" 


+ " = no solution. 


Immune " 


heated 


+ " = no solution. 


Immune " 


" 


+ normal serum + bacteria = solution. 



From the chart it is clear that there are two sub- 
stances concerned in bacteriolysis, one of which is found 
in any fresh serum, but is easily destroyed, and is called 
the complement. The other substance is found only in 
the immune serum, is relatively stable, and is known as 
the immune body or amboceptor. 

In hemolysis we find an analogy to bacteriolysis. 
Let a rabbit be immunized to sheep's blood-corpuscles. 
Now, if washed sheep's blood-corpuscles be subjected to 
the action of fresh serum from this rabbit, a speedy so- 
lution of the red cells ensues. If this serum is allowed 
to stand for several days, or is heated one-half hour to 
56° C, it will completely lose its solvent power. Now, 
the addition of a fresh normal serum, even of another 
species, will reactivate the heated or old serum. The 
following chart will indicate these reactions: 

Hemolysis. 
Rabbit serum, immune -f- corpuscles (sheep's) = solution. 

Rabbit " " heated + " " = no solution. 

Normal " + " '' = no solution. 

Rabbit " " heated + normal serum + corpuscles (sheep's) 

= solution. 

In hemolysis, as in bacteriolysis, besides the antigen 
(substance giving rise to amboceptors or antibodies) there 
are two substances. One of these is specific, /. e., only 
found in immune serum, and reacting only with the sub- 
stance used in producing the immune serum. This sub- 



SERUM REACTIONS 267 

stance is relatively stable, and is known as the amboceptor. 
The other substance is found in any serum, is absolutely 
non-specific, is easily destroyed, and is called the com- 
plement. In neither case will the amboceptor nor the 
complement acting alone cause a solution of the antigen. 

There are three substances necessary to bacteriolysis 
and hemolysis. To produce bacteriolysis there must be 
the specific antigen (as cholera vibrio in Pfeififer's phe- 
nomenon), the immune serum containing the amboceptor, 
and a complement. These three substances comprise 
the bacteriolytic system. Likewise, in hemolysis there 
is the red blood-cell, the amboceptor, and a complement, 
which comprise the hemolytic system. 

It will be noted that there is one factor common to 
both systems, viz., the complement. It will be evident 
that if we place in a test-tube a complete bacteriolytic 
system, with just enough complement to cause solution 
of the bacteria, and place this for a sufficient time in 
the optimum temperature for bacteriolysis, and then add 
two elements of the hemolytic system (amboceptor and 
blood-cells), no hemolysis will ensue, because all of the 
complement was used by the bacteriolytic system. 

Bordet and Gengou in 1901 showed that it was pos- 
sible to utilize this fact in the diagnosis of certain bac- 
terial infections. For instance, the heated serum of a 
suspected typhoid case plus typhoid bacilli is added 
to a serum containing complement (fresh guinea-pig 
serum) and incubated one hour. If this suspected serum 
contains typhoid amboceptors, there will have been 
a combination effected between the three elements of 
the bacteriolytic system, so that there will be no free 
complement left. If no amboceptor is present, all of 



268 



THE BLOOD 



the complement will remain unattached. Now, in 
order to show whether this complement has been fixed 
or deviated, two elements of a hemolytic system are 
added — namely, amboceptor and red corpuscles, and if 
the complement is fixed, no hemolysis can ensue. 

This is easily understood from a study of the swinging 
pendulum diagram, in which the complement is repre- 
sented in the pendulum. 



DACTERIOLYTIC 
SYSTEM 



HEMOL>/TI.C 
SYSTEM 




Fig. I02. — Pendulum diagram illustrating hemolysis.' 

This principle of "complement deviation" having been 
utilized in the diagnosis of infectious diseases, it occurred 
to Wassermann in 1906 to apply it to the diagnosis of 
syphilis. The antigen first used was the extract of a fetal 
syphilitic liver, but subsequent work has shown that the 
same reaction may be obtained with normal liver or 
spleen tissue, or with certain lipoid substances, and in 
this sense is not a true antigen-antibody reaction. The 
antibodies in the syphilitic blood, however, are specific. 
These are analogous to the bacteriolytic amboceptor of 
the pendulum diagram. 

* Not original, but unable to place credit where due. 



SERUM REACTIONS 269 

Technic of the Wassennann Test. — ^The following reagents 
are necessary: 

Antigen. — Extract of fetal syphilitic or normal liver, 
diluted I : 10. 

Antibodies (Analogous to Bacteriolytic Antibodies). — The 
serum or spinal fluid of the suspected patient. As controls, 
the serum of a syphilitic known to contain antibodies and the 
serum of a normal person known not to contain antibodies. 

Complement. — Fresh guinea-pig serum. Other fresh nor- 
mal sera may be used. This is diluted i : 10. 

Hemolytic Amboceptor. — The serum of a rabbit which has 
been immunized to sheep's red blood-corpuscles. This serum 
is inactivated before use by heating to 56° C. one-half hour, 
and diluted i : 1000 before using. 

Corpuscle Suspension. — Sheep's blood is defibrinated, 
washed three times with normal salt solution, and then 
diluted with normal salt solution to make a 5 per cent, sus- 
pension. 

In using these various reagents it is necessary to know that 
they are potent and of the proper strength, that is, to establish 
the titre of the reagent. This being determined, we are now 
ready for the Wassermann test, as carried out in the table 
on page 270. 

In the luetic control tube there will occur a combination 
between the antibodies in the serum and the antigen, which 
together will cause a fixation of the complement, so that 
when later the two elements of the hemolytic system are 
added, no hemolysis will occur. This inhibition of hemolysis 
indicates a positive syphilitic reaction. 

Control tube No. 3 is used to show that there is nothing 
in a normal serum which can effect this combination and 
deviation. Tube No. 4 shows that the patient's serum alone 
is not anticomplementary. Tube No. 5 shows that the 
hemolytic system is effective. Tube No. 6 shows that the 



270 



THE BLOOD 






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SERUM REACTIONS 27 1 

antigen alone is not anticomplementary. Tube No, 7 is 
introduced to show that hemolysis will not occur in the ab- 
sence of the complement. 

Modifications. — Certain modifications of this test 
have been suggested, chief of which is the Noguchi test. 
This differs from the Wassermann mainly in that an anti- 
human hemolytic system is used instead of an anti- 
sheep, because, according to the author, there is an 
appreciable error in the Wassermann in that there is 
present " in human serum a variable amount of natural 
antisheep amboceptor " capable of so changing the 
results that with sera containing only a small amount 
of syphilitic antibody the result will be negative. 

The following reagents are used : 

1. Antihuman hemolytic amboceptor prepared by re- 
peated injections of a rabbit with washed human blood- 
corpuscles. 

2. Complement. Fresh guinea-pig serum. 

3. Antigen. Organ extracts or a solution of lecithin. 

4. I per cent, suspension of human blood-corpuscles. 

5. The suspected serum. 

6. A known syphilitic serum. 

7. A serum known not to contain syphilitic antibodies. 
With these reagents the procedure is very little different 

from that outlined for the Wassermann test. 

Noguchi has further simplified it for the small laboratory 
by drying the amboceptor serum on slips of filter-paper, 
which can be kept a considerable time. The same procedure 
can be carried out with the antigen. While at first similar 
complement slips were prepared, it is now known that fresh 
complement is indispensable. 



272 



THE BLOOD 



Value of Wassermann Test. — The reaction is positive 
in 95 to 98 per cent, of all cases with syphilitic manifes- 
tations. In the late cases only a very slight inhibition 
of hemolysis may be noted. This has given rise to 
considerable difficulty in the interpretation of results. 
Kaplan states that the report should read "negative" 
or " positive," with no report of the degree of inhibition. 

Butler obtains the following results: 

No. of Per cent. 

cases. positive. 

Controls, non-syphilitic 53 o 

Primary syphilitic 10 100 

Secondary syphilitic 36 95 

Tertiary syphiltic 31 94 

Latent cases 16 56 

Parasyphilis and visceral syphilis 55 76 

Total cases 201 

Kaplan, in a study of diseases of the nervous system, 
obtained the following results: In 249 cases of quiescent 
tabes the Wassermann reaction was positive in 44 per 
cent.; in 57 cases of active tabes, 88 per cent., and in 64 
cases of general paresis, 88 per cent. 

By the Noguchi method about 7 per cent, of non- 
syphilitic sera will cause inhibition of hemolysis, while 
with the Wassermann, in about 9 per cent, of known 
syphilitic sera, hemolysis will occur. For this reason in 
doubtful cases it is well to apply both methods. 

It is probable that a positive reaction almost always 
means active syphilis even without manifestations, but 
it is not absolutely specific for syphilis, for the reaction 
has been obtained in leprosy. Kaplan states that " old 
leprosy cases present a much more definitely positive 
reaction than cases of old syphiHs." Many workers 
believe that a positive reaction in a late case may only 



SERUM REACTIONS 273 

indicate that the patient has once had syphilis. Against 
this view stands the fact that in other infectious diseases 
antibodies diminish or entirely disappear a few months 
after the active infection, and that in latent cases the 
reaction may disappear under treatment. On the other 
hand, there are certain cases which are considered clinic- 
ally as cured, and have remained so for years, that will 
continue to give the positive reaction in spite of any 
treatment. 

In the application of the test to the diagnosis of dis- 
eases of the nervous system and viscera one should al- 
ways bear in mind the possibility of a dual pathologic 
process, and a positive test should not be allowed to 
entirely overshadow the clinical findings. 

Effect of Treatment. — The positive reaction fre- 
quently disappears after a short course of treatment 
with mercury. This may be permanent, or, after a 
variable length of time, the reaction may return. Some 
cases thoroughly treated persist in giving a positive 
reaction. In hereditary syphilis it is often impossible 
to get rid of the reaction. Because the reaction may 
return, it is always safer to make several tests before 
deciding that further treatment is not indicated. This 
suggests that while a positive reaction may be accepted 
as an indication of syphilis, a negative reaction obtained 
after treatment may not exclude syphilis. 

Following treatment with salvarsan {" 606 "), Noguchi, 
in a total of 102 cases, finds that the positive reaction 
becomes negative in 33.7 per cent. 

18 



274 THE BLOOD 

X. TESTS FOR RECOGNITION OF BLOOD 

1. Quaiac Test.— The technic of this test has been 
given (p. 125). It may be appHed directly to a suspected 
fluid or, better, to the ethereal extract. Add a few 
cubic centimeters of glacial acetic acid to about 10 c.c. 
of the fluid; shake thoroughly with an equal volume of 
ether; decant, and apply the test to the ether. In case 
of dried stains upon cloth, wood, etc., dissolve the stain 
in distilled water and test the water, or press a piece of 
moist blotting-paper against the stain, and touch the 
paper with drops of the guaiac and the turpentine suc- 
cessively. 

2. Teichmann's Test.— This depends upon the pro- 
duction of characteristic crystals of hemin. It is a sensi- 




Fig. 103. — Teichmann's hemin crystals (Jakob). 

tive test and, when positive, is absolute proof of the 
presence of blood. A number of substances — lime, fine 
sand, iron rust — interfere with production of the crys- 
tals; hence negative results are not always conclusive. 
Dissolve the suspected stain in a few drops of normal 



SPECIAL BLOOD PATHOLOGY 275 

salt solution upon a slide. If a liquid is to be tested, 
evaporate some of it upon a slide and dissolve the residue 
in a few drops of the salt solution. Let_dry, apply a 
cover-glass, and run glacial acetic acid underneath it. 
Heat mry gently until "bubbles begin to form, replacing 
the acid as it evaporates. Allow to cool slowly. When 
cool, replace the acid with water, and examine for 
hemin crystals with 16 mm. and 4 mm. objectives. The 
crystals are dark-brown rhombic plates, lying singly 
or in crosses, and easily recognized (Fig. 103). Failure 
to obtain them may be due to too great heat or too rapid 
cooling. If not obtained at first let the slide stand in a 
warm place, as upon a hot-water radiator, for an hour. 

XI. SPECIAL BLOOD PATHOLOGY 

The more conspicuous characteristics of the blood in 
various diseases have been mentioned in previous sec- 
tions. Although the great majority of blood changes are 
secondary, there are a few blood conditions in which the 
changes are so prominent, or the etiology so obscure, that 
they are commonly regarded as blood diseases. These 
will receive brief consideration here. 

A. Anemia 

This is a deficiency of hemoglobin, or red corpuscles, 
or both. It is either primary or secondary. The dis- 
tinction is based chiefly upon etiology, although each 
'type presents a more or less distinctive blood-picture. 
Secondary anemia is that which is symptomatic of some 
other pathologic condition. Primary anemia is that 
which progresses without apparent cause. 



276 THE BLOOD 

1. Secondary Anemia.— The more important condi- 
tions which produce secondary or symptomatic anemia 
are: 

(a) Poor nutrition, which usually accompanies unsani- 
tary conditions, poor and insufficient food, etc. 

{b) Acute infectious diseases, especially rheumatism 
and typhoid fever. The anemia is more conspicuous 
during convalescence. 

(c) Chronic Infectious Diseases. — Tuberculosis, mala- 
ria, syphilis, leprosy. 

{d) Chronic exhausting diseases, as heart disease, 
chronic nephritis, cirrhosis of the liver, and gastro- 
intestinal diseases, especially when associated with 
atrophy of gastric and duodenal glands. The last may 
give an extreme anemia, indistinguishable from perni- 
cious anemia. 

{e) Chronic poisoning, as from lead, arsenic, and 
phosphorus. 

(/) Hemorrhage. — Either repeated small hemorrhages, 
as from gastric cancer and ulcer, uterine fibroids, etc., 
or a single large one. 

{g) Malignant Tumors. — These affect the blood partly 
through repeated small hemorrhages, partly through 
toxic products, and partly through interference with 
nutrition. 

{h) Animal Parasites. — Some cause no appreciable 
change in the blood; others, like the hookworm and 
Dihothriocephalus latus, may produce a very severe 
anemia, almost identical with pernicious anemia. Ane- 
mia in these cases is probably due both to toxins and to 
abstraction of blood. 

The blood-picture varies with the grade of anemia. 



SPECIAL BLOOD PATHOLOGY 277 

Diminution of hemoglobin is the most characteristic 
feature. In mild cases it is slight, and is the only blood 
change to be noted. In very severe cases hemoglobin 
may fall to 15 per cent. Red corpuscles are diminished 
in all but very mild cases, while in the severest cases 
the red corpuscle count is sometimes below 2,000,000. 
The color-index is usually decreased. 

Although the number of leukocytes bears no relation 
to the anemia, leukocytosis is common, being due to the 
same cause. 

Stained films show no changes in very mild cases. In 
moderate cases variations in size and shape of the red 
cells and polychromatophilia occur. Very severe cases 
show the same changes to greater degree, with addition 
of basophilic degeneration and the presence of normo- 
blasts in small or moderate numbers. Megaloblasts 
in very small numbers have been encountered in ex- 
tremely severe cases. They are especially abundant 
and may even predominate over the normoblasts in 
dibothriocephalus infection. Blood-plaques are usually 
increased. 

2. Primary Anemia. — The commonly described vari- 
eties of primary anemia are pernicious anemia and chlo- 
rosis, but splenic anemia may also be mentioned under 
this head. 

(i) Progressive Pernicious Anemia. — It is frequently 
impossible to diagnose this disease from the blood ex- 
amination alone. Severe secondary anemia sometimes 
gives an identical picture. Remissions, in which the 
blood approaches the normal, are common. All the 
clinical data must, therefore, be considered. 

Hemoglobin and red corpuscles are always greatly 



278 



THE BLOOD 



diminished. In none of Cabot's 139 cases did the count 
exceed 2,500,000, the average being about 1,200,000. 
In more than two-thirds of the cases hemoglobin was 
reduced to less extent than the red corpuscles; the color- 
index was, therefore, high. A low color-index probably 
indicates a mild type of the disease. 




Fig. 104. — A, Normal blood; B, chlorosis; C, pernicious anemia. The plate shows 
the sharp contrast between cells rich in hemoglobin and the pale cells of chlorosis, and also 
the poikilocytes and marked variations in size noted in pernicious anemia. A normo- 
blast and megaloblast also appear. Stained smears (from Greene's "Medical Diagnosis"). 

The leukocyte count may be normal, but is commonly 
diminished to about 3000. The decrease affects chiefly 
the polymorphonuclear cells, so that the lymphocytes are 
relatively increased. In some cases a decided absolute 
increase of lymphocytes occurs. Polymorphonuclear 
leukocytosis, when present, is due to some complication. 



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Explanation of Plate IX 

Fig. I. — Preparation from an advanced case of progressive perni- 
cious anemia from unknown cause: a, Megaloblasts or gigantoblasts; the 
protoplasm shows marked polychromasia; b, stained granules in erjlhro- 
cytes with normally stained protoplasm; c and d, polychromatophilic 
degeneration; e, megalocytes; /, normocytes. 

Fig. 2. — Preparation from the same case taken somje time later 
whUe the patient was subjectively and objectively in perfect health: 
ii, Punctate erythrocytes with normal and anemic degenerated proto- 
plasm; b, polynuclear leukocj'te; c, normal red blood-corpuscles; d, 
somewhat enlarged erythrocytes. 

Fig. 3. — Series of cells from a case of severe progressive per- 
nicious anemia of unknown etiology; preparation made two days ante- 
mortem: a. Nucleated red blood-corpuscles characterized as normo- 
blasts by the intense staining of the nuclei; a' and a", karyokinetic 
figures in erythrocytes; the protoplasm finely punctate; b, beginning 
karyolysis in a megaloblast; c, erythroblasts with coarse granulation of 
the protoplasm; d, nuclear remains (?) and line granulation of the 
protoplasm; e and /, finely punctate red blood-corpuscles; g, megalocyte 
with two blue nuclei; nuclear remains (?) in the polychrome protoplasm. 

(Nothnagel-Lazarus. ) 



PLATE IX 










Fig. 1. 



r • 

Fig. 2. 







JB^- 



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f- • 



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Fig. S. 



SPECIAL BLOOD PATHOLOGY 279 

The red corpuscles show marked variation in size and 
shape (Plate IX and Fig. 104). There is a decided 
tendency to large oval forms, and despite the abundance 
of microcytes, the average size of the corpuscles is gen- 
erally strikingly increased. Polychromatophilia and 
basophilic degeneration are common. Nucleated red 
cells are always present, although in many instances care- 
ful search is required to find them. In the great majority 
of cases megaloblasts exceed normoblasts in number. 
This ratio constitutes one of the most important points 
in diagnosis, since it is practically unknown in other 
diseases. Blood-plaques are diminished. 

The rare and rapidly fatal anemia which has been 
described under the name of aplastic anemia is probably 
a variety of pernicious anemia. Absence of any attempt 
at blood regeneration explains the marked difference in 
the blood-picture. Red corpuscles and hemoglobin are 
rapidly diminished to an extreme degree. The color- 
index is normal or low. The leukocyte count is normal 
or low, with relative increase of lymphocytes. Stained 
smears show only slight variations in size, shape, and 
staining properties of the red cells. There are no megalo- 
blasts and few or no normoblasts. 

(2) Chlorosis. — The clinical symptoms furnish the 
most important data for diagnosis. The blood resembles 
that of secondary anemia in many respects. 

The most conspicuous feature is a decided decrease of 
hemoglobin (down to 30 or 40 per cent, in marked cases), 
accompanied by a slight decrease in number of red cor- 
puscles. The color-index is thus almost invariably low, 
the average being about 0.5. 

As in pernicious anemia, the leukocytes are normal or 



28o THE BLOOD 

decreased in number, with a relative increase of lympho- 
cytes. 

In contrast to pernicious anemia (and in some degree 
also to secondary anemia) the red cells are of nearly 
uniform size, are uniformly pale (Fig, 104), and their 
average diameter is somewhat less than normal. Changes 
in size, shape, and staining reactions occur only in severe 
cases. Erythroblasts are rarely present. The number 
of plaques is generally decreased. 

(3) Splenic Anemia. — This is an obscure form of 
anemia associated with great enlargement of the spleen. 
It is probably a distinct entity. There is decided decrease 
of hemoglobin and red corpuscles, with moderate leu- 
kopenia and relative lymphocytosis. Osier's 15 cases 
averaged 47 per cent, hemoglobin and 3,336,357 red cells. 
Stained films show notable irregularities in size, shape, 
and staining properties only in advanced cases. Erythro- 
blasts are uncommon. 

B. Leukemia 

Except in rare instances, diagnosis is easily made 
from the blood alone. Two types of the disease are 
commonly distinguished: the myelogenous and the lym- 
phatic. Atypical and intermediate forms are not un- 
common. Pseudoleukemia, because of its clinical sim- 
ilarity to lymphatic leukemia, is generally described 
along with leukemia. 

1. Myelogenous Leukemia (Plate X).— This is usu- 
ally a chronic disease, although acute cases have been 
described. 

Hemoglobin and red corpuscles show decided decrease. 
The color-index is moderately low. 



PLATE X 




Fig. I. — Blood in lymphatic leukemia; X 700. On the left, chronic form 
of the disease; on the right, acute form (courtesy of Dr. W. P. Harlow). 




Fig. 2. — Blood in splenomyelogenous leukemia. Wright's stain. X 700 
(photographs by the author). 



SPEaAL BLOOD PATHOLOGY 28 1 

Most striking is the immense increase in number of 
leukocytes. The count in ordinary cases varies between 
100,000 and 300,000. Counts over 1,000,000 have been 
met. During remissions, the leukocyte count may fall 
to normal. 

While these enormous leukocyte counts are equaled in 
no other disease, and approached only in lymphatic 
leukemia and extremely high-grade leukocytosis, the 
diagnosis, particularly during remissions, depends more 
upon qualitative than quantitative changes. Although 
all varieties are increased, the characteristic and con- 
spicuous cell is the myelocyte. This cell never appears 
in normal blood; extremely rarely in leukocytosis; and 
never abundantly in lymphatic leukemia. In myelog- 
enous leukemia myelocytes usually constitute more than 
20 per cent, of all leukocytes. Da Costa's lowest case 
gave 7 per cent. The neutrophihc form is generally 
much more abundant than the eosinophiUc. Both show 
considerable variations in size. Very constant also is a 
marked absolute, and often a relative, increase of eosin- 
ophiles and basophiles. Polymorphonuclear neutro- 
philes and lymphocytes are relatively decreased. 

The red cells show the changes characteristic of a 
severe secondary anemia, except that nucleated reds are 
commonly abundant; in fact, no other disease gives so 
many. They are chiefly of the normoblastic type. 
Megaloblasts are uncommon. Blood-plaques are gen- 
erally increased. 

2. Lymphatic Leukemia (Plate X).— This form may 
be either acute or chronic. There is marked loss of 
hemoglobin and red corpuscles. The color-index is 
usually moderately low. 



282 THE BLOOD 

The leukocyte count is high, but lower than in the 
myelogenous type. Counts of 100,000 are about the 
average, but in many cases are much lower. This high 
count is referable almost wholly to increase of lympho- 
cytes. They generally exceed 90 per cent, of the total 
number. In chronic cases they are chiefly of the small 
variety; in acute cases, of the large form. Myelocytes 
are rare. 

The red corpuscles show the changes usual in severe 
secondary anemia. Erythroblasts are seldom abundant. 
Blood-plaques are decreased. 

3. Pseudoleukemia (Hodgkin's disease) resembles 
lymphatic leukemia in that there is marked and pro- 
gressive enlargement of the lymph-nodes. There is, 
however, no distinctive blood-picture. The changes in 
hemoglobin and red cells resemble those of a moderate 
symptomatic anemia, with rather low color-index. The 
leukocytes are commonly normal in number and relative 
proportions. 

4. Anaemia Infantum PseudoJeukaemica. — Under 
this name von Jaksch described a rare disease of infancy, 
the proper classification of which is uncertain. There is 
enlargement of liver and spleen, and sometimes of lymph- 
nodes, together with the following blood changes: grave 
anemia with deformed and degenerated red cells and 
many erythroblasts of both normoblastic and megalo- 
blastic types; great increase in number of leukocytes 
(20,000 to 100,000) and great variations in size, shape, 
and staining of leukocytes, with many atypic forms, and 
a few myelocytes. 

The table on the following page contrasts the distinct- 
ive blood-changes in the more common conditions. 



SPECIAI. BLOOD PATHOLOGY 283 



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CHAPTER IV 

THE STOMACH 

Laboratory methods may be applied to the diagnosis 
of stomach disorders in : I. Examination of the gastric con- 
tents removed with the stomach-tube. II. Certain other 
examinations which give information as to the condition 
of the stomach. 

I. EXAMINATION OF THE GASTRIC CONTENTS 

Stomach digestion consists mainly in the action of pep- 
sin upon proteins in the presence of hydrochloric acid 
and in the curdling of milk by rennin. The fat-splitting 
ferment, lipase, of the gastric juice has very little activity 
under normal conditions of acidity. 

Pepsin and rennin are secreted by the gastric glands 
as zymogens — pepsinogen and renninogen respectively 
— which are converted into pepsin and rennin by hydro- 
chloric acid. Hydrochloric acid is secreted by certain 
cells of the fundus glands. It at once combines loosely 
with the proteins of the food, forming acid-metaprotein, 
the first step in protein digestion. Hydrochloric acid, 
which is thus loosely combined with proteins, is called 
"combined" hydrochloric acid. The acid which is se- 
creted after the proteins present have all been converted 
into acid-metaprotein remains as "free" hydrochloric 
acid, and, together with pepsin, continues the process of 
digestion. 

284 



EXAMINATION OF THE GASTRIC CONTENTS 285 

At the height of digestion the stomach-contents consist 
essentially of: (i) Water; (2) free hydrochloric acid; 
(3) combined hydrochloric acid; (4) pepsin; (5) rennin; 
(6) mineral salts, chiefly acid phosphates, of no clinical 
importance; (7) particles of undigested and partly di- 
gested food; (8) various products of digestion in solution. 
In pathologic conditions there may be present, in addi- 
tion, various microscopic structures and certain organic 
acids, of which lactic acid is most important. 

A routine examination is conveniently carried out in the 
following order: 

(i) Give the patient a test-meal upon an empty stomach, 
washing the stomach previously if necessary. 

(2) At the height of digestion, usually in one hour, remove 
the contents of the stomach with a stomach-tube. 

(3) Measure and examine macroscopicaliy. 

(4) Filter. A suction filter is desirable, and may be neces- 
sary when much mucus is present. 

(5) During filtration, examine microscopically and make 
qualitative tests for — (a) free acids; (b) free hydrochloric 
acid; (c) lactic acid. 

(6) When sufficient filtrate is obtained, make quantitative 
estimations of — (a) total acidity; (b) free hydrochloric acid; 
(c) combined hydrochloric acid (if ne'cessary). 

(7) Make whatever additional tests seem desirable, as for 
blood, pepsin, or rennin. 

A. Obtaining the Contents 

■ Gastric juice is secreted continuously, but quantities 
suflSciently large for examination are not usually obtain- 
able from the fasting stomach. In clinical work, there- 
fore, it is desirable to stimulate secretion with food — 



286 THE STOMACH 

which is the natural and most efficient stimulus — before 
attempting to collect the gastric fluid. Different foods 
stimulate secretion to different degrees, hence for the 
sake of uniform results certain standard "test-meals" 
have been adopted. Those mentioned here give prac- 
tically the same results. 

1 . Test=meals. — It is customary to give the test-meal 
in the morning, since the stomach is most apt to be 
empty at that time. If it be suspected that the stomach 
will not be empty, it should be washed out with water 
the evening before. 

(i) Ewald's test-breakfast consists of a roll (or two 
slices of bread), without butter, and two small cups (300 
to 400 c.c.) of water, or weak tea, without cream or sugar. 
It should be well masticated. The contents of the 
stomach are to be removed one hour afterward, counting 
from the beginning, not the end of the meal. This 
test-meal has long been used for routine examinations. 
Its disadvantage is that it introduces, with the bread, a 
variable amount of lactic acid and numerous yeast-cells. 
This source of error may be eliminated by substituting 
a shredded whole-wheat biscuit for the roll. The 
shredded wheat test-meal is now widely used and is 
probably the most satisfactory for general purposes. 

(2) Boas' test-breakfast consists of a tablespoonful 
of rolled oats in a quart of water, boiled to one pint, with 
a pinch of salt added. It should be withdrawn in forty- 
five minutes to one hour. This meal does not contain 
lactic acid, and is usually given when the detection of 
lactic acid is important, as in suspected gastric cancer. 
The stomach should always be washed with water the 
evening previous. 



EXAMINATION OF THE GASTRIC CONTENTS 287 

2. Withdrawal of the Contents. — The Boas stomach- 
tube, with bulb, is probably the most satisfactory form. 
It should be of rather large caHber, and have an opening in 
the tip and one or two in the side near the tip. When not 
in use it should be kept in a vessel of borax solution, and 
should be well washed in hot water both before and after 
using. 

It is important confidently to assure the patient that 
introduction of the tube cannot possibly harm him; and 
that, if he can control the spasm of his throat, he will 
experience very little choking sensation. When patients 
are very nervous it is well to spray the throat with cocain 
solution. 

The tube should be dipped in warm water just before 
using: the use of glycerin or other lubricant is undesir- 
able. With the patient seated upon a chair, his cloth- 
ing protected by towels or a large apron, and his head 
tilted forward, the tip of the tube, held as one would a 
pen, is introduced far back into the pharynx. He is then 
urged to swallow, and the tube is pushed boldly into the 
esophagus until the ring upon it reaches the incisor teeth, 
thus indicating that the tip is in the stomach. If, now, 
the patient cough or strain as if at stool, the contents of 
the stomach will usually be forced out through the tube. 
Should it fail, the fluid can generally be pumped out by 
alternate compression of the tube and the bulb. If 
unsuccessful at first, the attempts should be repeated 
with the tube pushed a little further in, or withdrawn a 
few inches, since the distance to the stomach is not the 
same in all cases. The tube may become clogged with 
pieces of food, in which case it must be withdrawn, 
cleaned, and reintroduced. If, after all efforts, no fluid 



288 THE STOMACH 

is obtained, another test-meal should be given and with- 
drawn in forty-five minutes. 

As the tube is removed, it should be pinched between 
the fingers so as to save any fluid that may be in it. 

The stomach-tube must be used with great care, or not 
at all, in cases of gastric ulcer, aneurysm, uncompensated 
heart disease, and marked arteriosclerosis. Except in 
gastric ulcer, the danger lies in the retching produced, and 
the tube can safely be used if the patient takes it easily. 

B. Physical Examination 
Under normal conditions 30 to 50 c.c. of fluid can be 
obtained one hour after administering Ewald's breakfast. 
More than 60 c.c. points to motor insufficiency; less 
than 20 c.c, to too rapid emptying of the stomach, or 
else to incomplete removal. Upon standing, it separates 
into two layers, the lower consisting of particles of food, 
the upper of an almost clear, faintly yellow fluid. The 
extent to which digestion has taken place can be roughly 
judged from the appearance of the food-particles. 

The reaction is frankly acid in health and in nearly all 
pathologic conditions. It may be neutral or slightly 
alkaline in some cases of gastric cancer and marked 
chronic gastritis, or when contaminated by a consider- 
able amount of saliva. 

A small amount of mucus is present normally. Large 
amounts, when the gastric contents are obtained with 
the tube and not vomited, point to chronic gastritis. 
Mucus is recognized from its characteristic slimy appear- 
ance when the fluid is poured from one vessel into another. 
It is more frequently seen in stomach washings than in 
the fluid removed after a test-meal. 



EXAMINATION OF THE GASTRIC CONTENTS 289 

A trace of bile may be present as a result of excessive 
straining while the tube is in the stomach. Large 
amounts are very farely found, and generally point to 
obstruction in the duodenum. Bile produces a yellowish 
or greenish discoloration of the fluid. 

Blood is often recognized by simple inspection, but 
more frequently requires a chemic test. It is bright red 
when very fresh, and dark, resembling coffee-grounds, 
when older. Vomiting of blood, or hematemesis, may be 
mistaken for pulmonary hemorrhage, or hemoptysis. In 
the former the fluid is acid in reaction and usually dark 
red or brown in color and clotted, while in hemoptysis 
it is brighter red, frothy, alkaline, and usually mixed 
with a variable amount of mucus. 

Particles of food eaten hours or even days previously 
may be found, and indicate deficient motor power. 

Search should always be made for bits of tissue from 
the gastric mucous membrane or new growths. These, 
when examined by a pathologist, will sometimes render 
the diagnosis clear. 

C. Chemic Examination 
A routine chemic examination of the gastric contents 
involves qualitative tests for free acids, free hydrochloric 
acid, and organic acids, and quantitative estimations of 
total acidity, free hydrochloric acid, and sometimes 
combined hydrochloric acid. Other tests are applied 
when indicated. 

* I. Qualitative Tests.— (i) Free Acids.— The pres- 
ence or absence of free acids, without reference to the 
kind, is easily determined by means of Congo-red, 
although the test is not much used in practice. 

19 



290 THE STOMACH 

Congo-red Test. — Take a few drops of a strong alcoholic 
solution of Congo-red in a test-tube, dilute with water to a 
strong red color, and add a few cubic centimeters of filtered 
gastric juice. The appearance of a blue color shows the 
presence of some free acid (Plate XI, B, B'). Since the test 
is more sensitive to mineral than to organic acids, a marked 
reaction points to the presence of free hydrochloric acid. 

Thick filter-paper soaked in Congo-red solution, dried, and 
cut into strips may be used, but the test is much less delicate 
when thus applied. 

(2) Free Hydrochloric Acid. — In addition to its diges- 
tive function, free hydrochloric acid is an efficient anti- 
septic. It prevents or retards fermentation and lactic- 
acid formation, and is an important means of protection 
against the entrance of pathogenic organisms into the 
body. It is never absent in health. 

Amidobenzol Test. — To a little of the filtered gastric juice 
in a test-tube, or to several drops in a porcelain dish, add a 
drop of 0.5 per cent, alcoholic solution of dimethylamido- 
azobenzol. In the presence of free hydrochloric acid there 
will at once appear a cherry-red color, varying in intensity 
with the amount of acid (Plate XII, C). This test is very 
delicate; but, unfortunately, organic acids, when present in 
large amounts (above 0.5 per cent.), give a similar reaction. 

Boas' Test. — This test is less delicate than the preceding, 
but is more reliable, since it reacts only to free hydrochloric 
acid. 

In a porcelain dish mix a few drops of the gastric juice and 
the reagent, and slowly evaporate to dryness over a flame, 
taking care not to scorch. The appearance of a rose-red color, 
which fades upon cooling, shows the presence of free hydro- 
chloric acid (Plate XI, i). 



PLATE XI 






A, Uffelmann's reagent; A', A after the addition of gastric fluid 
containing lactic acid; B, water to which three drops of Congo-red 
solution have been added; B', change induced in B when gastric fluid 
containing free hydrochloric acid is added (Boston). 





I, Resorcin-test for free hydrochloric acid; 2, Glinzburg's test for hydro- 
chloric acid (Boston). 



EXAMINATION OF THE GASTRIC CONTENTS 29 1 

Boas' reagent consists of 5 gm. resublimed resorcinol, and 
3 gm. cane-sugar, in 100 c.c. alcohol. The solution keeps 
well, which, from the practitioner's view-point, makes it 
preferable to Giinzburg's phloroglucin-vanillin reagent (phlo- 
roglucin, 2 gm.; vanillin, i gm.; absolute alcohol, 30 c.c). 
The latter is just as delicate, is applied in the same way, and 
gives a sharper reaction (Plate XI, 2), but is unstable. 

(3) Organic Acids. — Lactic acid is the most common, 
and is taken as the type of the organic acids which appear 
in the stomach-contents. It is a product of bacterial 
activity. Acetic and butyric acids are sometimes 
present. Their formation is closely connected with that 
of lactic acid, and they are rarely tested for. When 
abundant, they may be recognized by their odor upon 
heating. 

Lactic acid is never present at the height of digestion 
in health. Although usually present early in digestion, 
it disappears when free hydrochloric acid begins to 
appear. Small amounts may be introduced with the 
food. Pathologically, small amounts may be present 
whenever there is stagnation of the gastric contents with 
deficient hydrochloric acid, as in many cases of dilatation 
of the stomach and chronic gastritis. The presence of 
notable amounts of lactic acid (more than o.i per cent, 
by Strauss' test) is strongly suggestive of gastric can- 
cer, and is probably the most valuable single symp- 
tom of the disease. 

As already stated, the Ewald test-breakfast introduces 
a small amount of lactic acid, but rarely enough to re- 
spond to the tests given here. In every case, however, 
in which its detection is important, the shredded- wheat 
biscuit or Boas' test-breakfast should be given, the 



292 THE STOMACH 

stomach having been thoroughly washed the evening 
before. 

Ufifelmann's Test for Lactic Acid. — Thoroughly shake 
up 5 c.c. of filtered stomach fluid with 50 c.c. of ether for at 
least ten minutes. Collect the ether and evaporate over a 
water-bath. Dissolve the residue in 5 c.c. water and test 
with Ufifelmann's reagent as follows: 

In a test-tube mix 3 drops concentrated solution of 
phenol and 3 drops saturated aqueous solution of ferric 
chlorid. Add water until the mixture assumes an amethyst- 
blue color. To this add the solution to be tested. The 
appearance of a canary-yellow color indicates the presence of 
lactic acid (Plate XI, A, A'). 

Uffelmann's test may be applied directly to the stomach- 
contents without extracting with ether, but is then neither 
sensitive nor reliable, because of the phosphates, sugars, and 
other interfering substances which may be present. 

Kelling's Test {Simofis Modification). — This is much more 
satisfactory than Ufifelmann's. To a test-tube of distilled 
water add sufficient ferric chlorid solution to give a faint 
yellowish tinge. Pour half of this into a second test-tube to 
serve as a control. To the other add a small amount of the 
gastric juice. Lactic acid gives a distinct yellow color which 
is readily recognized by comparison with the control. 

Strauss' Test for Lactic Acid. — This is a good test for 
clinical work, since it gives a rough idea of the quantity 
present and is not sufficiently sensitive to respond to the 
traces of lactic acid which some test-meals introduce. Strauss' 
instrument (Fig. 105) is essentially a separating funnel with 
a mark at 5 c.c. and one at 25 c.c. Fill to the 5 c.c. mark 
with filtered stomach fluid, and to the 25 c.c. mark with ether. 
Shake thoroughly for ten or fifteen minutes, let stand until the 
ether separates, and then, by opening the stop-cock, allow the 
liquid to run out to the 5 c.c. mark. Fill to the 25 c.c. mark 



EXAMINATION OF THE GASTRIC CONTENTS 



293 



with water, and add two drops of tincture of ferric chlorid 
diluted I : ID. Shake gently. If o.i per cent, or more lactic 
acid be present, the water will assume a 
strong greenish-yellow color. A slight 
tinge will appear with 0.05 per cent. 

(4) Pepsin and Pepsinogen. — Pep- 
sinogen itself has no digestive power. 
It is secreted by the gastric glands, and 
is transformed into pepsin by the ac- 
tion of a free acid. Although pepsin 
digests proteins best in the presence 
of free hydrochloric acid, it has a slight 
digestive activity in the presence of 
organic or combined hydrochloric acids. 

The amount is not influenced by 
neuroses or circulatory disturbances. 
Absence or marked diminution, there- 
fore, indicates organic disease of the 
stomach. It is an important point in 
diagnosis between functional and or- 
ganic conditions. Pepsin is rarely or never absent in 
the presence of free hydrochloric acid. 



Fig. 105. — Separatory 
funnel for Strauss' lac- 
tic acid test (Sahli). 



Test for Pepsin and Pepsinogen. — With a cork-borer cut 
small cylinders from the coagulated white of an egg, and cut 
these into discs of uniform size. The egg should be cooked 
very slowly, preferably over a water-bath, so that the white 
may be readily digestible. The discs may be preserved in 
glycerin, but must be washed in water before using. 

Place a disc in each of three test-tubes. 

Into tube No. i put 10 c.c. distilled water, 5 grains pepsin, 
U. S. P., and 3 drops of the official dilute hydrochloric acid. 



294 THE STOMACH 

Into tube No. 2 put 10 c.c. filtered gastric juice. 

Into tube No. 3 put 10 c.c. filtered gastric jUice and 3 drops 
dilute hydrochloric acid. 

Place the tubes in an incubator or warm water for three 
hours or longer. At intervals, observe the extent to which 
the egg-albumen has been digested. This is recognized by 
the depth to which the disc has become translucent. 

Tube No. I is used for comparison, and should show the 
effect of normal gastric juice. 

Digestion of the egg in tube No. 2 indicates the presence of 
both pepsin and free hydrochloric acid. 

When digestion fails in tube No. 2 and occurs in No. 3, 
pepsinogen is present, having been transformed into pepsin by 
the hydrochloric acid added. Should digestion fail in, this 
tube, both pepsin and pepsinogen are absent. 

(5) Rennin and Renninogen. — Rennin is the milk- 
curdling ferment of the gastric juice. It is derived from 
reiminogen through the action of hydrochloric acid. 
Lime salts also possess the power of transforming rennin- 
ogen into the active ferment. 

Deficiency of rennin has the same significance as 
deficiency of pepsin, and is more easily recognized. 
Since the two enzj-mes are almost invariably present or 
absent together, the test for rennin serves also as a test 
for pepsin. 

Test for Rennin. — Neutralize 5 c.c. filtered gastric juice 
with very dilute sodium hydroxid solution; add 5 c.c. fresh 
milk, and place in an incubator or in a vessel of water at 
about 104° F. Coagulation of the milk in ten to fifteen 
minutes shows a normal amount of rennin. Delayed coagu- 
lation denotes a less amount. 



EXAMINATION OF THE GASTRIC CONTENTS 295 

Test for Renninogen. — To 5 c.c. neutralized gastric Juice 
add 2 c.c. of I per cent, calcium chlorid solution and 5 c.c. 
fresh milk, and place in an incubator. If coagulation occurs, 
renninogen is present. 

(6) Blood. — Blood is present in the vomitus in a 
great variety of conditions. When found in the fluid 
removed after a test-meal, it commonly points toward 
ulcer or carcinoma. Blood can be detected in nearly 
one-half of the cases of gastric cancer. The presence of 
swallowed blood must be excluded. 

Test for Blood in Stomach-contents. — To 10 c.c. of the 

fluid add a few cubic centimeters of glacial acetic acid and 
shake the mixture thoroughly with an equal volume of ether. 
Separate the ether and apply to it the guaiac test (p. 125) ; or 
evaporate and apply the hemin test (p. 274) to the residue. 
When brown particles are present in the fluid, the hemin test 
should be applied directly to them. 

2. Quantitative Tests.— (i) Total Acidity.— The acid- 
reacting substances which contribute to the total acidity 
are free hydrochloric acid, combined hydrochloric acid, 
acid salts, mostly phosphates, and, in some pathologic 
conditions, the organic acids. The total acidity is 
normally about 50 to 75 degrees (see method below) or, 
when estimated as hydrochloric acid, about 0.2 to 0.3 
per cent. 

Tbpfer's Method for Total Acidity. — In an evaporating 
dish or small beaker (an " after-dinner " coffee-cup is a very 
convenient substitute) take 10 c.c. filtered stomach-contents 
and add three or four drops of the indicator, a i per cent, 
alcoholic solution of phenolphthalein. When the quantity of 



296 THE STOMACH 

stomach fluid is small, 5 c.c. may be used, but results are less 
accurate than with a larger amount. Add decinormal solution 
of sodium hydroxid drop by drop from a buret, until the fluid 
assumes a rose-red color which does not become deeper upon 
addition of another drop (Plate XII, A, A'). When this point 
is reached, all the acid has been neutralized. The end reaction 
will be sharper if the fluid be saturated with sodium chlorid. 
A sheet of white paper beneath the beaker facilitates recog- 
nition of the color change. 

In clinical work the amount of acidity is expressed by the 
number of cubic centimeters of the decinormal sodium hy- 
droxid solution which would be required to neutralize 100 c.c. 
of the gastric juice, each cubic centimeter representing one 
deforce of acidity. Hence multi]:)ly the number of cubic centi- 
meters of decinormal solution required to neutralize the 10 c.c. 
of stomach fluid by ten. This gives the number of degrees of 
acidity. The amount may be expressed in terms of hydro- 
chloric acid, if one remember that each degree is equivalent to 
0.00365 per cent, hydrochloric acid. Some one suggests that 
this is the number of days in the year, the last figure, 5, in- 
dicating the number of decimal places. 

Example. — Suppose that 7 c.c. of decinormal solution were 
required to bring about the end reaction in 10 c.c. gastric 
juice; then 7 X 10 = 70 deforces of acidity; and, expressed in 
terms of hydrochloric acid, 70 X 0.00365 = 0.255 P^'' c^^'- 

Preparation of decinormal solution is described in text- 
books on chemistry. The practitioner will find it best to 
have them made by a chemist, or to purchase from a chemic 
supply house. Preparation of an approximately decinormal 
solution is described on page 436. 

(2) Hydrochloric Acid. — After the Ewald and Boas 
test-breakfasts the amount of free hydrochloric acid 
varies normally between 25 and 50 degrees, or about o.i 



EXAMINATION OF THE GASTRIC CONTENTS 297 

to 0.2 per cent. In disease it may go considerably 
higher or may be absent altogether. 

When the amount of free hydrochloric acid is normal, 
organic disease of the stomach probably does not exist. 

Increase of free hydrochloric acid above 50 degrees 
(hyperchlorhydria) generally indicates a neurosis, but also 
occurs in most cases of gastric ulcer and beginning 
chronic gastritis. 

Decrease of free hydrochloric acid below 25 degrees 
{hy pochlorhydria) occurs in some neuroses, chronic gas- 
tritis, early carcinoma, and most conditions associated 
with general systemic depression. Marked variation in 
the amount at successive examinations strongly suggests 
a neurosis. Too low values are often obtained at the 
first examination, the patient's dread of the introduction 
of the tube probably inhibiting secretion. 

Absence of free hydrochloric acid (achlorhydria) occurs 
in most cases of gastric cancer and far-advanced chronic 
gastritis, in many cases of pernicious anemia, and some- 
times in hysteria and pulmonary tuberculosis. 

The presence of free hydrochloric acid presupposes a 
normal amount of combined hydrochloric acid, hence the 
combined need not be estimated when the free acid has 
been found. When, however, free hydrochloric acid is 
absent, it is important to know whether any acid is 
secreted, and an estimation of the combined acid then 
becomes of great value. The normal average after an 
Ewald breakfast is about 10 to 15 degrees, the quantity 
depending upon the amount of protein in the test-meal. 

Topfer's Method for Free Hydrochloric Acid. — In a 

beaker take 10 c.c. filtered stomach fluid and add 4 drops 



298 THE STOMACH 

of the indicator, a 0.5 per cent, alcoholic solution of dimethyl- 
amido-azobenzol. A red color instantly appears if free hydro- 
chloric acid be present. Add decinormal sodium hydroxid 
solution, drop by drop from a buret, until the last trace of red 
just disappears, and a canary-yellow color takes its place (Plate 
XII, C, C')- Read off the number of cubic centimeters of 
decinormal solution added, and calculate the degrees, or 
percentage of free hydrochloric acid, as in Topfer's method 
for total acidity. 

When it is impossible to obtain sufficient fluid for all the 
tests, it will be found convenient to estimate the free hydro- 
chloric acid and total acidity in the same portion. After 
finding the free hydrochloric acid as just described, add 4 
drops phenolphthalein solution, and continue the titration. 
The amount of decinormal solution used in both titrations 
indicates the total acidity. 

Topfer's Method for Combined Hydrochloric Acid. — 
In a beaker take 10 c.c. filtered gastric juice and add 4 
drops of the indicator, a i per cent, aqueous solution of sodium 
alizarin sulphonate. Titrate with decinormal sodium hy- 
droxid until the appearance of a bluish-violet color which does 
not become deeper upon addition of another drop (Plate XII, 
B, B'). It is difficult, without practice, to determine when 
the right color has been reached. A reddish- violet appears 
first. The shade which denotes the end reaction can be 
produced by adding 2 or 3 drops of the indicator to 5 c.c. 
of I per cent, sodium carbonate solution. 

Calculate the number of cubic centimeters of decinormal 
solution which would be required for 100 c.c. of stomach fluid. 
This gives, in degrees, all the acidity except the combined hydro- 
chloric acid. The combined hydrochloric acid is then found 
by deducting this amount from the total acidity, which has 
been previously determined. 

Example. — Suppose that 5 c.c. of decinormal solution were 
required to produce the purple color in 10 c.c. gastric juice; 



PLATE XII 



fv_ 



r 





^ 



ffl 



v 



K- 



< 



A, Gastric fluid to which a i per cent, solution of phenolphthalein 
has been added; B, gastric fluid to which a i per cent, solution of alizarin 
has been added; C, gastric fluid to which a 0.5 per cent, solution of 
dimethylamido-azobenzol has been added; A', .'\ after titration with a 
decinormal solution of sodium hydroxid; B', B after titration with a 
decinormal solution of sodium hydroxid; C, C after titration with a 
decinormal solution of sodium hydroxid (Boston). 



EXAMINATION OF THE GASTRIC CONTENTS 299 

then 5 X 10 = 50 = all the acidity except combined hydrochloric 
acid. Suppose, now, that the total acidity has already been 
found to be 70 degrees; then 70— 50= 20 degrees of combined 
hydrochloric acid; and 20X0.00365 = 0.073 per cent. 

When free hydrochloric acid is absent, it is probably 
more helpful to estimate the acid deficit than the com- 
bined hydrochloric acid. The acid deficit shows how 
far the acid secreted by the stomach falls short of satu- 
rating the protein (and bases) of the meal. It repre- 
sents the amount of hydrochloric acid which must be 
added to the fluid before a test for free hydrochloric 
acid can be obtained. It is determined by titrating 
with hydrochloric acid, using dimethyl-amido-azo- 

benzol as indicator, until the fluid assumes a red color. 
The amount of deficit is expressed by the number of 
cubic centimeters of the decinormal solution required 
for 100 c.c. of the stomach fluid. 

(3) Organic Acids. — There is no simple direct quan- 
titative method. After the total acidity has been deter- 
mined, organic acids may be removed from another 
portion of the gastric filtrate by shaking thoroughly 
with an equal volume of neutral ether, allowing the 
fluids to separate, and repeating this process until the 
gastric fluid has been extracted with eight or ten times 
its volume of ether. The total acidity is then deter- 
mined, and the difference between the two determinations 
indicates the amount of organic acids. 

(4) Pepsin. — No direct method is avaliable. The 
following is sufficient for clinical purposes: 

Hammerschlag's Method. — To the white of an egg add 
twelve times its volume of 0.4 per cent, hydrochloric acid 



300 THE STOMACH 

(dilute hydrochloric acid, U. S. P., 4 c.c; water, 96 c.c), 
mix well, and filter. This gives a i per cent, egg-albumen 
solution. Take 10 c.c. of this solution in each of three tubes 
or beakers. To .1 add 5 c.c. gastric juice; to B^ 5 c.c. water 
with 0.5 gm. pepsin; to C, 5 c.c. water only. Place in an 
incubator for an hour and then determine the amount of 
albumin in each mixture by Esbach's method. Tube C shows 
the amount of albumin in the test-solution. The difference 
betwetnC and B indicates the amountof albumin which would 
be digested by normal gastric juice. The difference between 
C and .1 gives the albumin which is digested by the fluid 
under examination. Schiitz has shown that the amounts of 
pepsin in two fluids are proportionate to the squares of the 
products of digestion. Thus, if the amounts of albumin di- 
gested in tubes A and B are to each other as 2 is to 4, the 
amounts of pepsin are to each other as 4 is to 16. 

Certain sources of error can be eliminated by diluting the 
gastric juice several times before testing. The most import- 
ant of these are that the law of Schiitz holds good only for 
comparatively dilute solutions, and that the products of 
peptic activity inhibit digestion. 

Mett's method is generally preferred to the preceding. 
Put three or four Mett's tubes about 2 cm. long into a small 
beaker with diluted gastric juice (i c.c. of the filtrate plus 15 
c.c. twentieth-normal hydrochloric acid). Place in an incu- 
bator for twenty-four hours, and then measure as accurately 
as possible the column which has been digested, using a milli- 
meter scale and a hand lens or, better, a low power of the 
microscope and an eye-piece micrometer. Square the aver- 
age length of this column (law of Schiitz) and multiply by 
the degree of dilution, 16. The maximum figure obtained in 
this way is 256, representing a digested column of 4 mm. 

Prepare Mett's tubes as follows: 

Beat up slightly the whites of one or two eggs and filter. 
Pour into a wide test-tube and stand in this a number of 



EXAMINATION OF THE GASTRIC CONTENTS 



301 



^'r>/./ 



capillary glass tubes, i to £; mm. 'kT ^ia^neter. 'IVJj^n the 
tubes are filled, plug their ends -vti^JV )>r/e^4 crumbs, /a,pd coag- 
ulate the albumin by heating in water jtistsKort of b6lli/igf,//r. 
Dip the ends of the tube in melted paraffin and preserv^^n^tjl , ^ 
needed. Bubbles, if present, will probably disappear in a " 
few days. When wanted for use, cut the tubes into lengths 
of about 2 cm. Discard any in which the albumin has sep- 
arated from the wall. 



D. Microscopic Examination 

A drop of unfiltered stomach-contents is placed upon 
a slide, covered with a cover-glass, and examined with 
the 16 mm. and 4 mm. objectives. A drop of Lugol's 







Fig. io6.^GeneraI view of the gastric contents: a. Squamous epithelial cells from 
esophagus knd mouth; b, leukocytes; c, cylindric epithelial cells; d, muscle-fibers; e, fat- 
droplets and fat-crystals; /, starch-granules; g, chlorophyl-containing vegetable mat- 
ters; k, vegetable spirals; i, bacteria; k, sarcinae; I, budding (yeast) fungi (Jakob). 



solution allowed to run under the cover will aid in dis- 
tinguishing the various structures. 

Under normal conditions little is to be seen except 
■ great numbers of starch-granules, with an occasional 



302 THE STOMACH 

epithelial cell, yeast-cell, or bacterium. Starch-granules 
are recogni^d by their concentric striations and the fact 
that they stain blue with iodin solutions when undi- 
gested, and reddish, due to erythrodextrin, when partially 
digested. 

Pathologically, remnants of food from previous meals, 
red blood-corpuscles, pus-cells, sarcinae, and excessive 
numbers of yeast-cells and bacteria may be encountered 
(Fig. io6). 

Remnants of food from previous meals indicate 
deficient gastric motility. 

Red Blood-corpuscles. — Blood is best recognized by 
the chemic tests already given. The corpuscles some- 
times retain a fairly normal appearance, but are generally 
so degenerated that only granular pigment is left. When 
only a few fresh looking corpuscles are present, they 
usually come from irritation of the mucous membrane 
by the tube. 

Pus-cells. — Pus is rarely encountered in the fluid 
removed after a test-meal. Considerable numbers of 
pus-corpuscles have been found in some cases of gastric 
cancer. Swallowed sputum must always be considered. 

Sarcinae. — These are small spheres arranged in cuboid 
groups, often compared to bales of cotton. They fre- 
quently form large clumps and are easily recognized. 
They stain brown with iodin solution. They signify fer- 
mentation. Their presence is strong evidence against 
the existence of gastric cancer, in which disease they 
rarely occur. 

Yeast-cells. — As already stated, a few yeast-cells 
may be found under normal conditions. The presence 
of considerable numbers is e\adence of fermentation. 



EXAMINATION OF THE GASTRIC CONTENTS 303 

Their appearance has been described (p. 171). They 
stain brown with iodin solution. 

Bacteria. — Numerous bacteria may be encountered, 
especially in the absence of free hydrochloric acid. The 
Boas-Oppler bacillus is the only one of special significance. 
It occurs in the majority of cases of cancer, and is rarely 
found in other conditions. Carcinoma probably fur- 
nishes a favorable medium for its growth. 




Fig. 107. — Boas-Oppler bacillus from case of gastric cancer (Boston). 

These bacilli (Fig. 107) are large (5 to 10 [i long), 
non-motile, and usually arranged in clumps or end to 
end in zig-zag chains. They stain brown with iodin 
solution, which distinguishes them from Leptothrix buc- 
calis (p. 377), which is not infrequently found in stomach 
fluid. They also stain by Gram's method. They are 
easily seen with the 4 mm. objective in unstained prepa^ 
rations, but are best recognized with the oil lens, after 
drying some of the fluid upon a cover-glass, fixing, and 
staining with a simple bacterial stain or by Gram's 
method. 



304 THE STOMACH 

A few large non-motile bacilli are frequently seen ; they 
cannot be called Boas-Oppler bacilli unless they are 
numerous and show something of the typical arrange- 
ment. 

E. The Gastric Contents in Disease 

In the diagnosis of stomach disorders the practitioner 
must be cautioned against relying too much upon exam- 
inations of the stomach-contents. A first examination 
is especially unreliable. Even when repeated examina- 
tions are made, the laboratory findings must never be 
considered apart from the clinical signs. 

The more characteristic findings in certain disorders 
are suggested here. 

1 . Dilatation of the Stomach. — Evidences of retention 
and fermentation are the chief characteristics of this 
condition. Hydrochloric acid is commonly diminished. 
Pepsin may be normal or sfightly diminished. Lactic 
acid may be detected in small amounts, but is usually 
absent when the stomach has been washed before giving 
the test-meal. Both motility and absorptive power are 
deficient. The microscope commonly shows sarcinae, 
bacteria, and great numbers of yeast-cells. Remnants 
of food from previous meals can be detected with the 
naked eye or microscopically. 

2. Gastric Neuroses. — The findings are variable. 
Successive examinations may show normal, increased, 
or diminished hydrochloric acid, or even entire absence 
of the free acid. Pepsin is usually normal. 

In the neurosis characterized by continuous hyperse- 
cretion (gastrosuccorrhea) , 40 c.c. or more of gastric juice 
can be obtained from the fasting stomach. Should 



EXAMINATION OF THE GASTRIC CONTENTS 305 

the fluid contain food-particles, it is probably the result 
of retention, not hypersecretion. 

3. Chronic Gastritis. — Free hydrochloric acid may be 
increased in early cases. It is generally diminished in 
well-marked cases, and is often absent in advanced 
cases. Lactic acid is often present in traces, rarely 
in notable amount. Secretion of pepsin and rennin is 
always diminished in marked cases. Mucus is frequently 
present, and is very significant of the disease. Motility 
and absorption are generally deficient. Small fragments 
of mucous membrane may be found, and when examined 
by a pathologist, may occasionally establish the diagnosis. 

4. Achylia Qastrica (Atrophic Gastritis). — This con- 
dition may be a terminal stage of chronic gastritis. It is 
sometimes associated with the blood-picture of pernicious 
anemia. It gives a great decrease, and sometimes entire 
absence of hydrochloric acid and ferments. The total 
acidity may be as low as i or 2 degrees. Small amounts 
of lactic acid may be present. Absorption and motihty 
are usually not affected greatly. 

5. Gastric Carcinoma. — As far as the laboratory 
examination goes, the cardinal signs of this disease are 
absence of free hydrochloric acid and presence of lactic 
acid and of the Boas-Oppler bacillus. These findings 
are, however, by no means constant. 

It is probable that some substance is produced by the 
cancer which neutralizes the free hydrochloric acid, and 
thus causes it to disappear earlier than in other organic 
diseases of the stomach. 

The presence of lactic acid is the most suggestive single 
symptom of gastric cancer. In the great majority of 
cases its presence in notable amount (o.i per cent, by 
20 



3o6 THE STOMACH 

Strauss' method) after Boas' breakfast, the stomach 
having been washed the evening before, warrants a 
tentative diagnosis of mahgnancy. 

Carcinoma seems to furnish an especially favorable 
medium for the growth of the Boas-Oppler bacillus, hence 
this micro-organism is frequently present. 

Blood can be detected in the stomach fluid by the 
chemic tests in nearly one-half of the cases, and is more 
common when the new growth is situated at the pylorus. 
Blood is present in the stool in nearly every case. 

Evidences of retention and fermentation are the rule in 
pyloric cancer. Tumor particles are sometimes found 
late in the disease. 

6. Gastric Ulcer.— There is excess of free hydrochloric 
acid in about one-half of the cases. In other cases the 
acid is normal or diminished. Blood is often present. 
The diagnosis must be based largely upon the clinical 
symptoms, and where ulcer is strongly suspected, it is 
probably unwise to use the stomach-tube. 

IL ADDITIONAL EXAMINATIONS WHICH GIVE INFOR- 
MATION AS TO THE CONDITION OF THE STOMACH 

1 . Absorptive Power of the Stomach. — This is a very 
unimportant function, only a few substances being ab- 
sorbed in the stomach. It is delayed in most organic dis- 
eases of the stomach, especially in dilatation and carci- 
noma, but not in neuroses. The test has Uttle practical 
value. 

Give the patient, upon an empty stomach, a 3-grain cap- 
sule of potassium iodid with a glass of water, taking care 
that none of the drug adheres to the outside of the capsule. 



EXAinNATION AS TO THE CONDITION OF STOMACH 307 

At intervals test the saliva for iodids by moistening starch- 
paper with it and touching with yellow nitric acid. A blue 
color shows the presence of an iodid, and appears normally 
in ten to fifteen minutes after ingestion of the capsule. A 
longer time denotes delayed absorption. 

Starch paper is prepared by soaking filter-paper in boiled 
starch and drying. 

2. Motor Power of the Stomach. — This refers to the 
rapidity with which the stomach passes its contents on 
into the intestine^. It is very important : intestinal diges- 
tion can compensate for insufficient or absent stomach 
digestion only so long as the motor power is good. 

Motility is impaired to some extent in chronic gastritis. 
It is especially deficient in dilatation of the stomach due 
to atony of the gastric wall or to pyloric obstruction, 
either benign or maUgnant. It is increased in most con- 
ditions with h^perchlorhydria. 

The best evidence of deficient motor power is the 
detection of food in the stomach at a time when it should 
be empty, e. g., before breakfast in the morning. WTien 
more than 60 c.c. of fluid are obtained with the tube one 
hour after a Ewald breakfast, deficient motility may be 
inferred. 

Ewald's salol test is scarcely so reliable as the above. It 
depends upon the fact that salol is not absorbed until it 
reaches the intestines and is decomposed by the alkaline 
intestinal juices. 

The patient is given 15 grains of salol with a test-breakfast, 
and the urine, passed at intervals thereafter, is tested for 
salicyluric acid. A few drops of 10 per cent, ferric chlorid 
solution are added to a small quantity of the urine. A violet 



3o8 THE STOMACH 

color denotes the presence of salicyluric acid. It appears 
normally in sixty to seventy- live minutes after ingestion of the 
salol. A longer time indicates impaired motor power. 

3. To Determine Size and Position of Stomach. — 

After removing the test-meal, while the tube is still in 
place, force quick puffs of air into the stomach by com- 
pression of the bulb. The puflfs can be clearly heard with 
a stethoscope over the region of the stomach, and no- 
where else. 

If desired, the patient may be given a dram of sodium 
bicarbonate in solution, followed immediately by the 
same amount of tartaric acid, also in solution; or he may 
take the two parts of a seidlitz powder separately. The 
carbon dioxid evolved distends the stomach, and its 
outline can easily be determined by percussion. 

4. Sahli's Desmoid Test of Gastric Digestion. — Two 
pills, one containing o.i gram iodoform, the other 0.05 
gram methylene-blue, are wrapped in little bags made of 
thin sheets of rubber and tied with a string of catgut. 
The bags must be carefully folded and tied. For detailed 
directions the reader is referred to Sahli's book, Diag- 
nostic Methods. 

The patient swallows the two bags with the aid of a 
little water during the noon meal, and the urine is tested 
at intervals thereafter. According to Sahli, the catgut 
is digested by gastric juice and not by pancreatic or 
intestinal juices. If gastric digestion is normal, iodin 
and methylene-blue can be detected in the urine in the 
afternoon or evening of the same day. The reaction 
may occur when digestion is very poor, provided gastric 
motility is diminished, but it is then delayed. If the 



EXAMINATION AS TO THE CONDITION OP STOMACH 309 

reaction does not. appear, gastric digestion has not 
occurred. 

Methylene-blue is recognized in the urine by the green or 
blue color which it imparts. It is sometimes eliminated as 
a chromogen; and a little of the urine must be acidified with 
acetic acid and boiled to bring out the color. 

To detect the iodin, some of the urine is decolorized by 
gently heating and filtering through animal charcoal. To 
10 c.c. are then added i c.c. dilute sulphuric acid, and 0.5 
c.c. of a I per cent, solution of sodium nitrite and 2 c.c. of 
chloroform. Upon shaking, a rose color will be imparted to 
the chloroform if iodin be present. 



CHAPTER V 
THE FECES 

As commonly practised, an examination of the feces is 
limited to a search for intestinal parasites or their ova. 
Much of value can, however, be learned from other simple 
examinations, particularly a careful inspection. Anything 
approaching a complete analysis is, on the'other hand, a 
waste of time for the clinician. 

The normal stool is a mixture of — (a) water; (b) 
undigested and indigestible remnants of food, as starch- 
granules, particles of meat, plant-cells and fibers, etc.; 
(c) digested foods, carried out before absorption could 
take place; (d) products of the digestive tract, as altered 
bile-pigments, mucus, etc. ; (e) products of decomposition, 
as indol, skatol, fatty acids, and various gases; (/) epi- 
thelial cells shed from the wall of the intestinal canal; 
(g) harmless bacteria, which are always present in 
enormous numbers. 

Pathologically, we may find abnormal amounts of 
normal constituents, blood, pathogenic bacteria, animal 
parasites and their ova, and biliary and intestinal con- 
cretions. 

The stool to be examined should be passed into a clean 
vessel, without admixture of urine. The offensive odor 
can be partially overcome with turpentine or 5 per cent, 
phenol. When search for amebae is to be made, the 
vessel must be warm, and the stool kept warm until 
examined; naturally, no disinfectant can be used. 

310 



MACROSCOPIC EXAMINATION 3 II 

I. MACROSCOPIC EXAMINATION 

1. Quantity. — The amount varies greatly with diet 
and other factors. The average is about loo to 150 gm. 
in twenty-four hours. 

2. Frequency. — One or two stools in twenty-four hours 
may be considered normal, yet one in three or four days 
is not uncommon with healthy persons. The individual 
habit should be considered in every case. 

3. Form and Consistence. — Soft, mushy, or liquid 
stools follow cathartics and accompany diarrhea. Co- 
pious, purely serous discharges without fecal matter 
are significant of Asiatic cholera, although sometimes 
observed in other conditions. Hard stools accompany 
constipation. Rounded scybalous masses are common in 
habitual constipation, and indicate atony of the muscular 
coat of the intestines Flattened, ribbon-like stools re- 
sult from some obstruction in the rectum, generally a 
tumor or stricture from a healed ulcer, most commonly 
syphilitic. When bleeding piles are absent, blood- 
streaks upon such a stool point to carcinoma. 

4. Color. — The normal light or dark-brown color is due 
chiefly to hydrobilirubin, which is formed from biHrubin 
by reducing processes in the intestines, largely the result 
of bacterial activity. The stools of infants are yellow, 
owing partly to their milk diet and partly to the presence 
of unchanged bihrubin. 

Diet and drugs cause marked changes: milk, a light 
yellow color; cocoa and chocolate, dark gray; various 
fruits, reddish or black ; iron and bismuth, dark brown or 
black; hematoxylin, red, etc. 

Pathologically, the color is important. A golden yellow 



312 THE FECES 

is generally due to unchanged bilirubin. Green stools 
are not uncommon, especially in diarrheas of childhood. 
The color is due to biliverdin or, sometimes, to chromo- 
genic bacteria. Putty-colored or " acholic " stools occur 
when bile is deficient, either from obstruction to outflow 
or from deficient secretion. The color is due less to 
absence of bile-pigments than to presence of fat. Similar 
stools are common in conditions Hke tuberculous peri- 
tonitis, which interfere with absorption of fats, and in 
pancreatic disease. 

Notable amounts of blood produce tarry black stools 
when the source of the hemorrhage is the stomach or 
upper intestine, and a dark brown or bright red as the 
source is nearer the rectum. When diarrhea exists the 
color may be red, even if the source of the blood is high 
up. Red streaks of blood upon the outside of the stool 
are due to lesions of rectum or anus. 

5. Odor. — Products of decomposition, chiefly indol 
and skatol, are responsible for the normal ofifensive odor. 
A sour odor is normal for nursing infants, and is noted in 
mild diarrheas of older children. In the severe diarrheas 
of childhood a putrid odor is common. In adults, stools 
emitting a very foul stench are suggestive of malignant 
or s}T3hiUtic ulceration of the rectum or gangrenous 
dysentery. 

6. Mucus. — Excessive quantities of mucus are easily 
detected with the naked eye, and signify irritation or 
inflammation. When the mucus is small in amount and 
intimately mixed with the stool, the trouble is probably 
in the small intestine. Larger amounts, not well mixed 
with fecal matter, indicate inflammation of the large 
intestine. Stools composed almost wholly of mucus and 



MACROSCOPIC EXAMINATION 313 

streaked with blood are the rule in dysentery, ileocolitis, 
and intussusception. 

In the so-called mucous colic or membranous enteritis 
shreds and ribbons of altered mucus, sometimes represent- 
ing complete casts of portions of the bowel, are passed. 
The mucus sometimes takes the form of frog-spawn-like 
masses. In some cases it is passed at variable intervals, 
with cohc; in others, with every stool, with only vague 
pains and discomfort. It is distinguished from inflam- 
matory mucus by absence of pus-corpuscles. The con- 
dition is not uncommon and should be more frequently 
recognized. It is probably a secretory neurosis, hence 
the name "membranous enteritis" is inappropriate. 

7. Concretions. — Gall-stones are probably more com- 
mon than is generally supposed, and should be searched 
for in every case of obscure colicky abdominal pain. 
Intestinal concretions (enteroliths) are rare. Intestinal 
sand, consisting of sand-like grains, is especially common 
in neurotic conditions, such as mucous colitis. 

Concretions can be found by breaking up the fecal 
matter in a sieve (which may be improvised from gauze) 
while pouring water over it. It must be remembered that 
gall-stones, if soft, may go to pieces in the bowel. 

8. Animal Parasites. — Segments of tapeworms and 
the adults and larvae of other parasites are often found in 
the stool. They are best searched for in the manner 
described for concretions. The search should be pre- 
ceded by a vermicide and a brisk purge. Patients fre- 
quently mistake vegetable tissue (long fibers from poorly 
masticated celery or " greens," cells from oranges, etc.) 
for intestinal parasites, and the writer has known 
physicians to make similar mistakes. Even slight famil- 



314 THE FECES 

iarity with the microscopic structure of vegetable tissue 
will prevent the chagrin of such errors. 

9. Curds. — The stools of nursing infants frequently 
contain whitish curd-like masses, due either to imperfect 
digestion of fat or casein or to excess of these in the diet. 
When composed of fat, the masses are soluble in ether, 
and give the Sudan III test. If composed of casein, 
they will become tough and fibrous-like when placed in 
formalin (10 per cent.) for twenty-four hours. 

11. CHEMIC EXAMINATION 

Complicated chemic examinations are of little value to 
the clinician. Certain tests are, however, important. 

1. Blood. — When present in large amount blood pro- 
duces such changes in the appearance of the stool that it 
is not likely to be overlooked. Traces of blood (occult 
hemorrhage) can be detected only by special tests. 
Recognition of occult hemorrhage has its greatest value 
in diagnosis of gastric cancer and ulcer. It is constantly 
present in practically every case of gastric cancer, and is 
always present, although usually intermittently, in ulcer. 
Traces of blood also accompany malignant disease of the 
bowel, the presence of certain intestinal parasites, and 
other conditions. 

Detection of Occult Hemorrhage. — Soften a portion of the 
stool with water, shake with an equal volume of ether to 
remove fat, and discard the ether. Treat the remaining 
material with about one-third its volume of glacial acetic 
acid and extract with ether. Should the ether not separate 
well, add a little alcohol. Apply the guaiac test to the ether 
as already described (p. 125). 



MICROSCOPIC EXAMINATION 315 

In every case iron-containing medicines must be stopped, 
and blood-pigment must be excluded from the food by giving 
an appropriate diet, e. g., bread, milk, eggs, and fruit. At the 
beginning of the restricted diet give a dram of powdered 
charcoal, or 7 grains of carmin, so as to mark the correspond- 
ing stool. 

2. Bile. — Normally, unaltered bile-pigment is never 
present in the feces of adults. In catarrhal conditions 
of the small intestine bilirubin may be carried through un- 
changed. It may be demonstrated by filtering (after 
mixing with water if the stool be solid) and testing the 
filtrate by Gmelin's method, as described under The 
Urine. 

Hydrobilirubin will give a red color if a little of the 
stool be rubbed up with saturated mercuric chlorid 
solution and allowed to stand twenty-four hours. The 
red color is likewise imparted to microscopic structures 
which are stained with hydrobilirubin. A green color 
in this test shows the presence of unchanged bilirubin. 

III. MICROSCOPIC EXAMINATION 

Care must be exercised in selection of portions for 
examination. A random search will often reveal nothing 
of interest. A small bit of the stool, or any suspicious- 
looking particle, is placed upon a slide, softened with 
water if necessary, and pressed out into a thin layer with 
a cover-glass. A large slide — about 2 by 3 inches — 
with a correspondingly large cover will be found conve- 
nient. Most of the structures which it is desired to see 
can be found with a 16 mm. objective. Details of struc- 
ture must be studied with a higher power. 



3i6 



THE FECES 



The bulk of the stool consists of granular debris. 
Among the recognizable structures met in normal and 
pathologic conditions are: Remnants of food, epithelial 
cells, pus-corpuscles, red blood-corpuscles, crystals, bac- 
teria, and ova of animal parasites (Fig. io8). 

I . Remnants of Food. — These include a great variety 
of structures which are very confusing to the student. 
Considerable study of normal feces is necessary for their 
recognition. 






mm 








Fig. io8. — Microscopic elements of normal feces: a. Muscle-fibers; b, connective 
tissue; c, epithelial cells; d, white blood -corpuscles; e. spiral vessels of plants; f-h, vege- 
table cells; J, plant hairs; k, triple phosphate crystals; /, stone cells. Scattered among 
these elements are micro-organisms and debris (after v. Jaksch). 



Vegetable fibers are generalh' recognized from their 
spiral structure or their pits, dots, or reticulate mark- 
ings; vegetable cells, from their double contour and the 
chlorophyl bodies which many of them contain. These 
cells are apt to be mistaken for the ova of parasites. 
Starch-granules sometimes retain their original form, but 
are ordinarily not to be recognized except by their stain- 
ing reaction. They strike a blue color with Lugol's solu- 



MICROSCOPIC EXAMINATION 



317 



tion when undigested; a red color, when slightly digested. 
Muscle-fibers are yellow, and when poorly digested appear 
as short, transversely striated cylinders with rather 
squarely broken ends (Fig. 109). Generally, the ends 
are rounded and the striations faint, or only irregularly 
round or oval yellow masses are found. Curds of milk 
are especially important in the stools of children. They 
must be distinguished from small masses of Jat (p. 314). 




Fig. log.- 



-Poorly digested muscle-fiber in feces showing striations (X 200) (photograph 
by the author). 



Excess of any of these structures may result from 
excessive ingestion or deficient intestinal digestion. 

2. Epithelial Cells. — A few cells derived from the 
wall of the alimentary canal are a constant finding. They 
show all stages of degeneration, and are often unrecog- 
nizable. A marked excess has its origin in a catarrhal 
condition of some part of the bowel. Squamous cells 
come from the anal orifice; otherwise the form of the 
cells gives no clue to the location of the lesion. 



3l8 THE FECES 

3. Pus. — Amounts of pus sufficient to be recognized 
with the eye alone indicate rupture of an abscess into 
the bowel. If well mixed with the stool, the source is 
high up, but in such cases the pus is apt to be more or 
less completely digested, and hence unrecognizable. 
Small amounts, detected only by the microscope, are 
present in catarrhal and ulcerative conditions of the in- 
testine, the number of pus-cells corresponding to the 
severity and extent of the process. 

4. Blood=corpuscles. — Unaltered red corpuscles are 
rarely found unless their source is near the anus. Ordi- 
narily, only masses of blood-pigment can be seen. Blood 
is best recognized by the chemic tests fp. 274). 

5. Bacteria. — In health, bacteria constitute about one- 
third of the weight of the dried stool. They are beneficial 
to the organism, although not actually necessary to its 
existence. It is both difficult and unprofitable to iden- 
tify them. The great majority belong to the colon 
bacillus group, and are negative to Gram's method of 
staining. 

In some pathologic conditions the character of the 
intestinal flora changes, so that Gram-staining bacteria 
very greatly predominate. As shown by R. Schmidt, 
of Neusser's clinic in Vienna, this change is most constant 
and most striking in cancer of the stomach, owing to 
large numbers of Boas-Oppler bacilli, and is of consider- 
able value in diagnosis. He believes that a diagnosis 
of gastric carcinoma should be very unwillingly made 
with an exclusively "Gram-negative" stool, while a 
"Gram-positive" stool, due to bacilli (which should also 
stain brown with Lugol's solution), may be taken as very 
strong evidence of cancer. A Gram-positive stool due 



MICROSCOPIC EXAMINATION 319 

to cocci is suggestive of intestinal ulceration. The 
technic is the same as when Gram's method is applied 
to other material (p. 409), except that the smear is fixed 
by immersion in methyl-alcohol for five minutes instead 
of by heat. Fuchsin is the best counterstain. The deep 
purple Gram-staining bacteria stand out much more 
prominently than the pale-red Gram-negative organisms, 
and one may be misled into thinking them more numer- 
ous even in cases in which they are much in the minority. 
The number of Boas-Oppler bacilli can be increased by 
administering a few ounces of sugar of milk the day 
before the examination. The bacteria can be obtained 
comparatively free from food remnants by mixing a 
little of the feces with water, allowing to settle for a 
short time, and making smears from the supernatant 
fluid. 

Owing to the difl&culty of excluding swallowed sputum, 
the presence of the tubercle bacillus is less significant in 
the feces than in other material. It may, however, be 
taken as evidence of intestinal tuberculosis when clinical 
signs indicate an intestinal lesion and reasonable care is 
exercised in regard to the sputum. Success in the 
search will depend largely upon careful selection of the 
portion examined. A random search will almost surely 
fail. Whitish or grayish flakes of mucus or blood- 
stained or purulent particles should be spread upon 
slides or covers and stained by the method given upon p. 
168. In the case of rectal ulcers, swabs can be made 
directly from the ulcerated surface. 

6. Crystals. — Various crystals may be found, but few 
have any significance. Slender, needle-like crystals of 
fatty acids and soaps (Fig. 36) and triple phosphate 



320 THE FECES 

crystals (Fig. io8) are common. Characteristic octahe- 
dral crystals of calcium oxalate (Fig. 51) appear after in- 
gestion of certain vegetables. Charcot-Leyden crystals 
(Fig. 9) are not infrequently encountered, and strongly 
suggest the presence of intestinal parasites. Yellowish 
or brown, needle-like or rhombic crystals of hematoidin 
(Fig. 36) may be seen after hemorrhage into the bowel. 
7. Parasites and Ova. — The stool should be well mixed 
with water and allowed to settle. The ova will be found 
in the upper or middle portions of the sediment. The 
flagellates are best found in the liquid stool after a dose 
of salts. Descriptions will be found in the following 
chapter. 

IV. FUNCTIONAL TESTS 

1. Schmidt's Test Diet. — Much can be learned of the 
various digestive functions from a microscopic study of 
the feces, especially when the patient is upon a known 
diet. For this purpose the standard diet of Schmidt is 
generally adopted. This consists of: 

Morning 0.5 liter milk and 50 gm. toast. 

Forenoon 0.5 liter porridge, made as follows: 40 gm. 

oatmeal, 10 gm. butter, 200 c.c. milk, 
300 c.c. water, and one egg. 

Midday 125 gm. hashed meat, with 20 gm. butter, 

fried so that the interior is quite rare; 
250 gm. potato, made by cooking 190 
gm. potato with 100 c.c. milk and 10 gm. 
butter, the whole boiled down to 250 c.c. 

Afternoon Same as morning. 

Evening Same as forenoon. 

At the beginning of the diet, the stool should be 
marked off with carmin or charcoal. One should famil- 



FUNCTIONAL TESTS 32 1 

iarize himself with the microscopic appearance of the 
feces of normal persons upon this diet. 

Deficiency of starch digestion is recognized by the 
number of starch-granules which strike a blue color with 
iodin. With exception of those inclosed in plant cells 
none are present normally. 

The degree of protein digestion is ascertained by the 
appearance of the muscle-fibers. Striations are clearly 
visible only when digestion is imperfect (Fig. 109). Ac- 
cording to Schmidt, the presence of nuclei in muscle-fibers 
denotes complete absence of pancreatic function. The 
presence of connective-tissue shreds indicates deficient 
gastric digestion, since raw connective tissue is digested 
only in the stomach. These shreds can be recognized 
macroscopically by examining in a thin layer against a 
black background, and microscopically by their fibrous 
structure and the fact that they clear up when treated 
with acetic acid. 

Digestion of fats is checked up by the amount of 
neutral fat. 

2. Sahli's Qlutoid Test, — The Schmidt test diet in- 
volves some inconvenience for the patient, and inter- 
pretation of results requires much experience upon the 
part of the physician. A number of other methods of 
testing the digestive functions have been proposed. The 
glutoid test of SahH is one of the most satisfactory. 
This is similar to his desmoid test of gastric digestion 
described on page 308. A glutoid capsule containing 
0.15 gram iodoform is taken with an Ewald breakfast. 
The capsule is not digested by the stomach fluid, but 
is readily digested by pancreatic juice. Appearance of 
iodin in the saUva and urine within four to six hours 
21 



322 THE FECES 

indicates normal gastric motility, normal intestinal di- 
gestion, and normal absorption. Instead of iodoform, 
0.5 gram salol may be used, salicyluric acid appearing 
in the urine in about the same time. For tests for iodin 
and salicyluric acid, see pages 307 and 309. 

The glutoid capsules are prepared by soaking gelatin 
capsules in formalin. Sahli states that filled capsules 
can be purchased of A. G. Haussmann, in St. Gall, 
Switzerland. 

3. Miiller's Test for Trypsin. — A calomel purge is 
given two hours after a meal. Particles of the feces are 
placed upon solidified blood-serum. This is incubated 
at a temperature of 55° to 60° C. to prevent action of 
bacteria. Digestion of the serum — indicated by a 
translucent, roughened, depressed surface — presumably 
shows the presence of trypsin, and indicates pancreatic 
sufficiency. Trypsin can seldom be detected without 
the preceding purge. 



CHAPTER VI 
ANIMAL PARASITES 

Animal parasites are common in all countries, but are 
especially abundant in the tropics, where, in some places, 
almost every native is host for one or more species. 
Because of our growing intercourse with these regions 
the subject is assuming increasing importance in this 
country. Many parasites, hitherto comparatively un- 
known here, will probably become more common. 

Some parasites produce no symptoms, even when 
present in large numbers. Others cause very serious 
symptoms. It is, however, impossible to make a sharp 
distinction between pathogenic and non-pathogenic 
varieties. Parasites which cause no apparent ill effects 
in one individual may, under certain conditions, produce 
marked disturbances in another. The disturbances are 
so varied, and frequently so indefinite, that diagnosis can 
rarely be made from the clinical symptoms. It must rest 
upon detection, by the naked eye or the microscope, of 
(a) the parasites themselves, (b) their ova or young 
progeny, or (c) some of their products. 

Unlike bacteria, the great majority of animal parasites 
-multiply by means of alternating and differently formed 
generations, which require widely different conditions 
for their development. The few exceptions are chiefly 
among the protozoa. Multiplication of parasites within 

323 



324 ANIMAL PARASITES 

the same host is thus prevented. In the case of the 
hook-worm, for example, there is no increase in the num- 
ber of worms in the host's intestine, except through re- 
infection from the outside. The young are carried out 
of the intestine and must pass a certain period of devel- 
opment in warm, moist earth before they can again 
enter the human body and grow to maturity. In general, 
this alternation of periods of development takes place in 
one of three ways: 

(i) The young remain within the original host, but 
travel to other organs, where they do not reach maturity, 
but lie quiescent until taken in by a new host. A good 
example is Trichinella spiralis. 

(2) The young or the ova which subsequently hatch 
pass out of the host, and either {a) go through a simple 
process of growth and development before entering 
another host, as is the case with the hook-worm, or {b) 
pass through one or more free-living generations, the 
progeny of which infect new hosts, as is the case with 
Strongyloides intestinalis. 

(3) The young or ova or certain specialized forms 
either directly {e. g., malarial parasites) or indirectly 
(e. g., tapeworms) reach a second host of different 
species, where a widely different process of development 
occurs. The host in which the adult or sexual existence 
is passed is called the 'definitive or final host; that in 
which the intermediate or larval stage occurs, the 
intermediate host. Man, for example, is the definitive 
host for TcEuia saginata, and the intermediate host for 
the malarial parasites and Tcenia echinococcus. 

A few words concerning the classification and nomen- 
clature of living organisms in general will be helpful 



ANIMAL PARASITES 325 

at this place. Individuals which are alike in all essential 
respects are classed together as a species. Closely related 
species are grouped together to form a genus; genera 
which have certain characteristics in common make up 
a family; families are grouped into orders; orders into 
classes; and classes, finally, into the branches or phyla, 
which make up the kingdom. In some cases these groups 
are subdivided into intermediate groups — subphyla, 
subfamilies, etc., and occasionally, slight differences 
warrant subdivision of the species into varieties. The 
animal kingdom comprises nine branches: Protozoa, 
Porifera, Ccelenterata, Echinodermata, Vermidea, Arth- 
ropoda, Mollusca, Prochordata, and Chordata. 

The scientific name of an animal or plant consists of 
two parts, both Latin or Latinized words, and is printed 
in italics. The first part is the name of the genus and 
begins with a capital letter; the second is the name of the 
species and begins with a lower case letter, even when it 
was originally a proper name. When there are varieties 
of a species, a third part, the designation of the variety, 
is appended. The author of the name is sometimes in- 
dicated in Roman type immediately after the name 
of the species. Examples: Spirochceta vincenti, often 
abbreviated to Sp. vincenti when the genus name has 
been used just previously; Staphylococcus pyogenes 
albus; Necator americanus, Stiles. 

At the present time there is great confusion in the 
.naming and classification of parasites. Some have been 
given a very large number of names by different observers, 
and in many cases different parasites have been described 
under the same name. The alternation of generations 
and the marked differences in some cases between male 



326 ANIMAL PARASITES 

and female have contributed to the confusion, different 
forms of the same parasite being described as totally 
unrelated species. 

The number of parasites which have been described 
as occurring in man and the animals is extremely large. 
Only those which are of medical interest are mentioned 
here. They belong to three phyla — Protozoa, Vermidea, 
and Arthropoda. 

PHYLUM PROTOZOA 

These are unicellular organisms, the simplest types 
of animal life. There is very little differentiation of 
structure. Each contains at least one, and some several 
nuclei. Some contain contractile vacuoles; some have 
cilia or flagella as special organs of locomotion. They 
reproduce by division, by budding, or by sporulation. 
Sometimes there is an alternation of generations, in one 
of which sexual processes appear, as is the case with the 
malarial parasites. The protozoa are very numerous, 
the subphylum Sarcodina alone including no less than 
5000 species. Most of the protozoa are microscopic in 
size ; a few are barely visible to the naked eye. One can 
gain a general idea of their appearance by examining 
water (together with a little of the sediment) from the 
bottom of any pond. Such water usually contains amebae 
and a considerable variety of ciliated and flagellated 
forms. 

The following is an outline of those protozoa which 
are of medical interest, together vvith the subphyla and 
classes to which they belong. 



PHYLUM PROTOZOA 



327 



PHYLUM PROTOZOA 

SuBPHYLUM I. SARCODINA. — Locomotion by means of pseudo- 
podia. 

Class Rhizopoda. — Pseudopodia form lobose or reticulose processes. 



Genus. 
Entamoeba. 



Species. 
E. histolytica. 
E. tetragena. 
E. coli. 
E. buccalis. 



StJBPHYLUM II. MASTIGOPHORA (FLAGELLATA).— Locomotion 
by means of flagella. 

Class Zoomastigophora. — Forms in which animal characteristics pre- 
dominate. 



Genus. 


species. 


Spirochaeta. 


Sp. obermeieri. 




Sp. vincenti. 




Sp. buccalis. 




Sp. dentium. 




Sp. refringens. 


Treponema. 


T. pallidum. 




T. pertenue. 


Trypanosoma. 


T. gambiense. 




T. cruzi. 




T. lewisi. 




T. evansi. 




T. brucei. 




T. equiperdum. 


Leishmania. 


L. donovani. 




L. tropica. 




L. infantum. 


Cercomonas. 


C. hominis. 


Bodo. 


B. urinarius. 


Trichomonas. 


T. vaginalis. 




T. intestinalis. 




T. pulmonalis. 


Lamblia. 


L. intestinalis. 



328 ANIMAL PARASITES 

SuBPHYLUM III. SPOROZOA.— All members parasitic. Propaga- 
tion by means of spores. No special organs of locomotion. 
Class Telosporidia.— Sporulation ends the life of the individual. 

Genus. Species. 

Coccidium. C. cuniculi. 

Plasmodium. P. vivax. 

P. malariae. 

P. falciparum. 
Babesia. B. bigeminum. 

SuBPHYLUM IV^ INFUSORIA.— Locomotion by means of cflia. 
Class Ciliata. — Cilia present throughout life. 

Getius. Species. 

Balantidium. B. coli. 



SUBPHYLUM SARCODINA 
Class Rhizopoda 

These are protozoa the body substance of which 
forms changeable protoplasmic processes, or pseudo- 
podia, for the taking in of food and for locomotion. 
They possess one or several nuclei. 

I. Genus Entamoeba. — (i) Entamoeba Histolytica. — 
This organism is found, often in large numbers, in the 
stools of tropical dysentery and in the pus and walls 
of hepatic abscesses associated with dysentery, and 
is generally regarded as the cause of the disease. It 
is a colorless, granular cell, 20 to 40 ^i in diameter 
(Fig. no). It contains one or more distinct vacuoles, 
a round nucleus, which ordinarily is obscured by the 
granules, and frequently red blood-corpuscles and bac- 
teria. When at rest its shape is spheric, but upon a 
warm slide it exhibits the characteristic ameboid motion, 
constantly changing its shape or moving slowly about. 
This motion is its most distinctive feature. If neutral 
red in 0.5 per cent, solution be run under the cover-glass, 



PHYLUM PROTOZOA 



329 



it will be taken up by the amebae and other protozoa 
and render them conspicuous without killing them 
("vital staining"). 

When the presence of amebae is suspected, the stool 
should be passed into a warm vessel and kept warm 
until and during the examination. A warm stage can 
be improvised from a plate of copper with a hole cut in 
the center. This is placed upon the stage of the mi- 
croscope, and one of the projecting ends is heated with 



i 




Fig. no. — Amoeba coli in intestinal mucus, with blood-corpuscles and bacteria (Losch). 



a small flame. Amebae are most likely to be found in 
grayish or blood-streaked particles of mucus. Favor- 
able material for examination can be obtained at one's 
convenience by inserting into the rectum a large catheter 
with roughly cut lateral openings. A sufficient amount 
of mucus or fecal matter will usually be brought away 
by it. 

(2) Other Entamebae. — Entamceba coli, a similar but 
somewhat smaller organism (10 to 20 fi), with less dis- 



33© ANIM.'VL PARASITES 

tinct pseudopodia and more distinct nucleus, has fre- 
quently been found in the stools of healthy persons. 
E. tetragena has recently been described. It apparently 
produces a chronic diarrhea and is not confined to the 
tropics. Another, E. buccalis, has been found in decay- 
ing teeth A number of similar organisms have been 
described as occurring in pus and in ascitic and other 
body fluids, but it is probable that in many cases, at 
least, the structures seen were ameboid body cells. 

SUBPHYLUM MASTIGOPHORA (FLAGELLATA) 
Qass Zodmastigophora 

The protozoa of this subphylum are provided with one 
or several whip-like appendages with lashing motion, 
termed flagella, which serve for locomotion and, in 
some cases, for feeding. They generally arise from the 
anterior part of the organism. Some members of the 
group also possess an undulating membrane — a delicate 
membranous fold which extends the length of the body, 
and somewhat suggests a fin. When in active motion 
this gives the impression of a row of cilia. The flagellata 
do not exhibit ameboid motion, and, in general, maintain 
an unchanging oval or spindle shape, and contain a single 
nucleus. The cytoplasm contains numerous granules 
and usually several vacuoles, one or more of which may 
be contractile. Encystment as a means of resisting 
unfavorable conditions is common. 

I. Genus Spirochaeta. — The spirochaetae appear to 
occupy a position midway between the bacteria and 
protozoa, but are more frequently described with the 
latter. 

(i) Spirochaeta Recurrentis. — This spirochaite was 



PHYLUM PROTOZOA 33 1 

described by Obermeier as the cause of relapsing fever. 
It appears in the circulating blood during the febrile 
attack, and, unlike the malarial parasite, lives in the 
plasma without attacking the red corpuscles. The 
organism is an actively motile spiral, 16 to 40 ;m long, 
with three to twelve wide, fairly regular turns. It can 
be seen in fresh unstained blood with a high dry lens, 
being located by the commotion which it creates among 




Fig. III. — Spirochaete of relapsing fever ( X looo) (Karg and Schmorl). 

the red cells. For diagnosis, thin films, stained with 
Wright's or some similar blood-stain, are used (Fig. iii). 

Besides Spirochceta recurrentis, a number of distinct 
strains have been described in connection with different 
types of relapsing fever: Sp. novyi (Plate VII), Sp. kochi, 
Sp. duUoni, and Sp. carteri. 

(2) Spirochaeta Vincenti. — In stained smears from the 
ulcers of Vincent's angina (p. 380) are found what 
appear to be two organisms. One, the " fusiform bacil- 



332 ANIMAL PARASITES 

lus/' is a slender rod, 6 to 12 /t/ long, pointed at both 
ends and sometimes curved. The other is a slender 
spiral organism, 30 to 40 fi long, with three to eleven 
comparatively shallow turns (Fig. 153). These were 
formerly thought to be bacteria, a spirillum and bacillus 
living in symbiosis. The present tendency is to regard 
them as stages or forms of the same organism, and to 
class them among the spirochaeta^. The same organisms 
are quite constantly present in large numbers in ulcera- 
tive stomatitis and in noma They are not infrequently 
found in small numbers in normal mouths. 




O 



Fig. 112. — Spiral organisms: A, Treponema pallidum; B, Spirochaeta refringens; C, 
Spirochaeta dentium. Two red corpuscles are also shown ( X 1200). 

(3) Other Spirochaetae. — A number of harmless forms 
are of interest because of the possibility of confusing 
them with the more important pathogenic varieties. 
Of these, Sp. buccalis and Sp. dentium are inhabitants 
of the normal mouth. The former is similar in morphol- 
ogy to Sp. vincenti. Sp. dentium (Fig. 112) is smaller, 
more delicate, has deep curves, and may be easily mis- 
taken for Treponema pallidum. It, also, stains reddish 
with Giemsa's stain. In suspected syphilitic sores of 
the mouth it is, therefore, important to make smears 



PHYLUM PROTOZOA 333 

from the tissue juices rather than from the surface 
(see p. 389). Sp. refringens is frequently present upon 
the surface of ulcers, especially about the genitals, and 
has doubtless many times been mistaken for Treponema 
pallidum. It can be avoided by properly securing the 
material for examination; but its morphology should 
be sufficient to prevent confusion. It is thicker than 
the organism of syphilis, stains more deeply, and has 
fewer and shallower curves (Fig. 112). Giemsa's stain 
gives it a bluish color. 

2. Genus Treponema.— (i) Treponema Pallidum. — 
This is the organism of syphilis. Its description and 
methods of diagnosis will be found on p. 388. 

(2) Treponema pertenue, morphologically very similar 
to Treponema pallidum, was found by Castellani in 
yaws, a skin disease of the tropics. 

3. Genus Trypanosoma. — Trypanosomes have been 
found in the blood-plasma of a great variety of verte- 
brates. Many of them appear to produce no symptoms, 
but a few are of great pathologic importance. As seen 
in the blood, they are elongated, spindle-shaped bodies, 
the average length of different species varying from 10 
to 70 f£. Along one side there runs a delicate undulating 
membrane, the free edge of which appears to be somewhat 
longer than the attached edge, thus throwing it into 
folds. Somewhere in the body, usually near the middle, 
is a comparatively pale-staining nucleus; and near the 
posterior end is a smaller, more deeply staining chromatin 
mass, the micronucleus or blepharoplast. A number of 
coarse, deeply staining granules, chromatophores, may 
be scattered through the cytoplasm. A flagellum arises 
in the blepharoplast, passes along the free edge of the 



334 ANIMAL PARASITES 

undulating membrane, and is continued anteriorly as a 
free flagellum. These details of structure are well 
shown in Plate VII. 

The life history of the trypanosomes is not well known. 
In most cases there is an alternation of hosts, various 
insects playing the part of definitive host. 

Trypanosomes have been much studied of late, and 
many species have been described. Of these, only a few 



> t 





/ 



Fig. 113. — Trypanosoma lewisi in blood of rat. The red corpuscles were decolorized 
with acetic acid (X 1000) (photograph by the author from a slide presented by Prof. 

Novy). 

have medical interest. At least two have been found 
in man. 

Trypanosoma gamhiense is the parasite of African 
" sleeping sickness." Its detection in the blood is 
described on p. 247. 

Trypanosoma cruzi is a small form which has been 
found in the blood of man in Brazil. 

Trypanosoma lewisi, a very common and apparently 
harmless parasite of gray rats, especially sewer rats, is 



PHYLUM PROTOZOA 335 

interesting because it closely resembles the pathogenic 
forms, and is easily obtained for study. Its posterior 
end is more pointed than that of T. gambiense. 

Trypanosoma evansi, T. brucei, and T. equiperdum 
produce respectively surra, nagana, and dourine, which 
are common and important diseases of horses, mules, 
and cattle in the Philippines, East India, and Africa. 

4. Genus Leishmania. — The several species which 
compose this genus are apparently closely related to the 
trypanosomes, but their exact classification is undeter- 
mined. They have been grown outside the body and 
their transformation into flagellated trypanosome-like 
structures has been demonstrated. Calkins places them 
in the genus Herpetomonas. 

(i) Leishmania donovani is the cause of kala-azar, an 
important and common disease of India. The " Leish- 
man-Donovan bodies " are round or oval structures, 
2 to 3 w in diameter, with two distinct chromatin masses, 
one large and pale, the other small and deeply staining. 
The parasites are especially abundant in the spleen, 
splenic puncture being resorted to for diagnosis. They 
are readily found in smears stained by any of the Roman- 
owsky methods. They lie chiefly within endothelial 
cells and leukocytes They are also present within 
leukocytes in the peripheral blood, but are difficult to 
find in blood-smears. 

(2) Leishmania tropica resembles the preceding. It 
is found, lying intracellularly, in the granulation tissue 
of Delhi boil or Oriental sore. 

(3) Leishmania infantum has been found in an obscure 
form of infantile splenomegaly in Algiers. 

5. Genus Cercomonas. — (i) Cerccmonas hominis has 



33^ 



ANIMAL PARASITES 



been found in the feces in a variety of diarrheal condi- 
tions, and in from lo to 25 per cent, of healthy persons in 
tropical regions. It is probably harmless. The body is 
10 to 12 ^ long, is pointed posteriorly, and has a flagel- 
lum at the anterior end (Fig. 114). The nucleus is 
difficult to make out. 



"^^^g^Vr^ 



Fig. 114. — Cercoraonas hominis (about X 500): A, Larger variety; B, smaller variety • 

(Davaine). 

6. Genus Bodo. — (i) Bodo urinarius is sometimes seen 
in the urine, darting about in various directions. It is 
probably an accidental contamination, or at most a 
harmless invader. It has a lancet-shaped body, about 
10 u long, and 4s somewhat twisted upon itself, with 
two flagella at the end. 





Fig. 115. — Trichomonas vaginalis (about X looo) (after Kolliker and Scanzoni). 

7. Genus Trichomonas.— (i) Trichomonas Vaginalis. 

— The acid discharge of catarrhal vaginitis sometimes 
contains this parasite in abundance. It is oval or pear- 
shaped, one to three times the diameter of a red blood- 
corpuscle in length, and has a cluster of flagella at one 
end (Fig. 115). As seen in fresh material it is not unlike 



PHYLUM PROTOZOA 337 

a pus-corpuscle in size and general appearance, but is 
actively motile. When in motion the flagella are not 
easily seen. No pathogenic significance is ascribed to 
it in the vagina, but a few cases have been reported in 
which it was apparently the cause of a urethritis in the 
male. This and similar organisms, such as cercomonas 





Fig. ii6. — Lamblia intestinalis from the intestines of a mouse (about X 2000) (Grass! 
and Schweiakofi). 

and bodo, might be mistaken for spermatozoa by the 
totally inexperienced worker. 

(2) Other Trichomonads. — Various forms have been 
described, regarded by some as identical with T. vagi- 
nalis, by others as distinct species. Among these are 
T. intestinalis, sometimes found in the feces in diarrheal 
conditions, and T. pulmonalis, which has been encoun- 
tered in the sputum of persons suffering from pulmonary 
gangrene and putrid bronchitis. 
22 



338 ANIMAL PARASITES 

8. Genus Lamblia. — (i) Lamblia intestinalis is a very 
common parasite in the tropics, but is generally consid- 
ered of little pathogenic importance. It is pear shaped, 
measures about 10 to 15 fJ., and has a depression on one 
side of the blunt end, by which it attaches itself to the 
tops of the epithelial cells of the intestinal wall. Three 
pairs of flagella are arranged about the depression and 
one pair at the pointed end (Fig. 116). 

SUBPHYLUM SPOROZOA 
Qass Telosporidia 

All the members of this class are parasitic, but only 
a few have been observed in man, and only one genus, 
Plasmodium, is of much importance in human pathology. 
Propagation is by means of spores, and sporulation ends 
the life of the individual. In some species there is an 
alternation of generations, in one of which sexual proc- 
esses appear. In such cases the male individual may 
be provided with flagella. Otherwise, there are no 
special organs of locomotion. 

I. Genus Coccidium. — (i) Coccidium cuniculi. — This 
is a very common parasite of the rabbit and has been 
much studied; but extremely few authentic cases of 
infection in man have been reported. The parasite, 
which when fully developed is ovoid in shape and 
measures about 30 to 50 u in length and has a shell- 
like integument, develops within the epithelial cells of 
the bile-passages. Upon reaching adult size it divides 
into a number of spores or merozoites which enter other 
epithelial cells and repeat the cycle. A sexual cycle 
outside the body, which suggests that of the malarial 
parasite, but does not require an insect host, also occurs. 



PHYLUM PROTOZOA 339 

Infection takes place from ingestion of the resulting 
sporozoites. 

2. Genus Plasmodium. — This genus includes the ma- 
larial parasites which have already been described (p. 248). 

3. Genus Babesia. — The proper position of this genus 
is uncertain. It is placed among the flagellates by some. 
The chief member is Babesia bigeminum, the cause of 
Texas fever in cattle. It is a minute, pear-shaped or- 
ganism, lying in pairs within the red blood-corpuscles. 
An organism, B. (or Piroplasma) hominis, described as 
occurring in the red cells in " tick-fever " of Montana, is 
also placed in this genus, but its pathogenicity and even 
its existence are questionable. 

SUBPHYLUM INFUSORU 
Qass Qliata 

The conspicuous feature of this class is the presence 
of cilia. These are hair-like appendages which have a 
regular to-and-fro motion, instead of the irregular lash- 
ing motion of flagella. They are also shorter and more 
numerous than flagella. Most infusoria are of fixed 
shape and contain two nuclei. Contractile and food- 
vacuoles are also present. Encystment is common. 
Only one species is of medical interest. Certain ciliated 
structures, which have been described as infusoria, 
notably in sputum and nasal mucus, were probably 
ciliated body cells. 

1. Genus Balantidium. — (i) Balantidium Coli. — This 
parasite, formerly called Paramcecium coli, is an occa- 
sional inhabitant of the colon of man, and sometimes 
produces diarrhea. It is an oval organism, about o.i mm. 
long, is covered with cilia, and contains a bean-shaped 



34° ANIMAL PAHASITES 

macronucleus, a globular micronucleus, two contractile 
vacuoles, and variously sized granules (Fig. 117). 




Fig. 117.— Balantidiumcoli (about X 300) (after Eichhorst). 

Its ordinary habitat is the rectum of the domestic 
pig, where it apparently causes no disturbance. It 
probably reaches man in the encysted condition. 

PHYLUM VERMIDEA 
Of the worms, many species are parasitic in man and 
the higher animals. In some cases man is the regular 
host; in others, the usual habitat is some one of the ani- 
mals, and the occurrence of the worm in man is more or 
less accidental. Such are called incidental parasites. 
Only those worms that are found in man with sufficient 
frequency to be of medical interest are mentioned here. 

PHAT.UM \^RMIDEA 
SUBPHYLUM I. PLATYHELMINTHES.— Flat-worms. 
Class Trematoda. — Flukes. Unsegmented, leaf shaped. 
Genus. Species. 

Fasciola. F. hepatica. 

Dicrocoelium. D. lanceatum. 

Opisthorchis. Op. felineus. 

Op. sinensis. 
Paragonimus. P. westermani. 

Schistosomum. S. haematobium. 

S. japonicum. 



PHYLUM VERMIDEA 



Class Cestoda. — Tapeworms. Segmented, ribbon shaped. 



341 



Genus. 


Species. 


Taenia. 


T. saginata. 




T. solium. 




T. echinococcus. 


Hymenolepis. 


H. nana. 


Dipylidium. 


D. caninum. 



Dibothriocephalus. D. latus. 

SuBPHYLUM II. NEMATHELMINTHES.— Round-worms. 
Class Nematoda. — Unsegmented, cylindric or fusiform. 



Genus. 


Species. 


Anguillula. 


A. aceti. 


Ascaris. 


A. lumbricoides. 


Oxyuris. 


0. vermicularis. 


Filaria. 


F. bancrofti. 




F. philippinensis. 




F. perstans. 




F. diuma. 




F. medinensis. 


Uncinaria. 


U. duodenalis. 


Necator. 


N. americanus. 


Strongyloides. 


S. intestinalis. 


Trichinella. 


T. spiralis. 


Trichocephalus. 


T. trichiuris. 



SUBPHYLUM PLATYHELMINTHES 
Class Trematoda 

The trematode worms, commonly known as " flukes," 
are flat, unsegmented, generally tongue- or leaf-shaped 
worms. They are comparatively small, most species 
averaging between 5 and 15 mm. in length. They pos- 
sess an incomplete digestive tract, without anus, and are 
provided with one or more sucking disks by means of 
which they can attach themselves to the host. Some 
are also provided with booklets. Nearly all species are 
hermaphroditic, and the eggs of nearly all are operculated 
(provided with a lid), the only important exception 



342 ANIMAL PARASITES 

being Schistosomum hematobium, the egg of which has a 
characteristic spine. Development takes place by al- 
ternation of generations, the intermediate generation 
occurring in some water animal: mollusks, amphibians, 
fishes, etc. 

1. Genus Fasciola. — (i) Fasciola Hepatica. — The 
" liver fluke " inhabits the bile-ducts of numerous herbiv- 
orous animals, especially sheep, where it is an important 
cause of disease. It brings about obstruction of the bile- 
passages, with enlargement and degeneration of the liver 




Fig. ii8. — Fasciola he[)atica, about two-thirds natural size (Mosler and Peiper). 

— " liver rot." A species of snail serves as intermediate 
host. The worm is leaf shaped, the average size being 
about 2.8 by 1.2 cm. The anterior end projects like a 
beak (head-cone 3 to 4 mm. long) (Fig. 118). Ova ap- 
pear in the feces. They are yellowish brown, oval, 
operculated, and measure about 0.13 by 0.07 mm. 

2. Genus Dicrocoelium. — (i)Dicrocoeliumlanceatum 
is often associated with the liver fluke in the bile-passages 
of animals, but is neither so common nor so widely 
distributed geographically. It has rarely been observed 
in man. It is smaller (length about i cm.) and more 



PHYLUM VERMIDEA 343 

elongated. The long diameter of the eggs is about 0.04 
mm. 

3. Genus Opisthorchis. — (i) Opisthorchis felineus 

inhabits the gall-bladder and bile-ducts of the domestic 
cat and a few other animals. Infection in man has 
been repeatedly observed in Europe, and especially in 
Siberia. The body is flat, yellowish-red in color, and 
almost transparent. It measures 8 to 11 mm. by 1.5 to 
2 mm. The eggs are oval, with a well-defined operculum 
at the narrower end, and contain a ciliated embryo when 
deposited. They measure about 30 by 11 fi. 

(2) Opisthorchis sinensis, Uke the preceding fluke, 
inhabits the gall-bladder and bile-ducts of domestic 
cats and dogs. It is, however, much more frequent in 
man, being a common and important parasite in certain 
parts of Japan and China. The number present may 
be very great; over 40CK) were counted in one case. 
The parasite resembles Op. felineus in shape and color. 
It is 10 to 14 mm. long and 2.5 to 4 mm. broad. The 
eggs have a sharply defined lid and measure 27 to 30 by 
15 to 17 /[/. When they appear in the feces they contain 
a ciliated embryo. The intermediate host is unknown. 

4. Genus Paragonimus. — (i) Paragonimus wester- 
mani, called the " lung fluke," is also a common parasite 
of man in Japan, China, and Korea. It is likewise found 
in dogs, cats, and pigs in these countries, and, according 
to Ward and Stiles, in North America also. It inhabits 
the lung, causing the formation of small cavities. Mod- 
erate hemoptysis is the principal symptom. Ova are 
readily found in the sputum; the worms themselves are 
seldom seen, except postmortem. The worms are faint 
reddish-brown in color, egg shaped with the ventral 



344 ANIMAL PARASITES 

surface flattened, and measure 8 to lo mm. by 4 to 6 mm. 
The ova, which are found in the sputum, are thin shelled, 
brownish yellow, and average about 0.093 by 0.057 n^™- 
Little is known of the development outside the body. 

5. Genus Schistosomum. — (i) Schistosomum Haema- 
tobium. — This trematode, frequently called Bilharzia 
hcematobia, is an extremely common cause of disease 
(bilharziasis or Egyptian hematuria) in northern Africa, 
particularly in Egypt. 

Unlike the other flukes, the sexes are separate. The 
male is 12 to 14 mm. long and i mm. broad. The body 
is flattened and the lateral edges curl ventrally, forming a 
longitudinal groove, in which the female lies (Fig. 119). 




Fig. iig. — Schistosomum hccmatobium, male and female (about X 4) with eggs (about 
X 70) (von Jaksch). 

The latter is cylindric in shape, about 20 mm. long and 
0.25 mm. in diameter. The eggs are an elongated oval, 
about 0.15 mm. long, yellowish in color, and slightly 
transparent. They possess no lid, such as characterize 
the eggs of most of the trematodes, but are provided 
with a thorn-like spine which is placed at one end or 
laterally near the end. 

In man the worm lives in the veins, particularly the 
portal vein and the veins of the bladder and rectum, lead- 
ing to obstruction and inflammation. The eggs penetrate 
into the tissues and are present in abundance in the 
mucosa of the bladder and rectum. They also appear in 
the urine and feces. The mode of infection is unknown. 



PHYLUM VERMIDEA 345 

(2) Schistosomum japonicum resembles the preceding 
morphologically, but both the male and female are 
smaller. The ova present no spines and somewhat re- 
semble those of Uncinaria duodenalis. It was discov- 
ered in Japan in 1904 and is apparently common in that 
country. It probably inhabits the arteries. 

Qass Cestoda 

The cestodes, or tapeworms, are very common para- 
sites of both man and the animals. In the adult stage 
they consist of a linear series of flat, rectangular segments 
(proglottides), at one end of which is a smaller segment, 
the scolex or head, especially adapted by means of suck- 
ing discs and booklets for attachment to the host. 
The series represents a colony, of which the scolex is 
ancestor. The proglottides are sexually complete in- 
dividuals (in most cases hermaphroditic), which are 
derived from the scolex by budding. With the excep- 
tion of the immature segments near the scolex, each 
contains a uterus filled with ova. 

The large tapeworms, Tcenia saginata, T. solium, and 
Dihothriocephalus latus, are distinguished from one an- 
other mainly by the structure of the scolex and the 
uterus. The scolex should be studied with a low- 
power objective or a hand lens. The uterus is best 
seen by pressing the segment out between two plates 
of glass. 

All the tapeworms pass a larval stage in the tissues of 
an intermediate host, which is rarely of the same species 
as that which harbors the adult worm. From the ova 
which have developed in the proglottides of the adult 
worm, and which pass out with the feces of the host, 



346 



ANIMAL PARASITES 



there develop embryos, or oncospheres, each provided 
with three pairs of horny hooklets. When the oncosphere 
is taken into the intestines of a suitable animal, it pene- 
trates to the muscles or viscera and there forms a cyst 
in which develop usually one, but sometimes many, 
scoHces, which are identical with the head of the adult 
worm. When the flesh containing this cystic stage is 
eaten without sufficient cooking to destroy the scolices, 
the latter attach themselves to the intestinal wall and 
produce adult tapeworms by budding. 



iglllllil"""^' iii"-iTr||iiiiiimin 




Fig. I20. — Taenia saginata (Eichhorst). 



Ordinarily, only the adult stage occurs in man. In the 
case of TcEfiia echinococcus only the larval stage is found. 
T. saginata and T. solium may infect man in either stage, 
although the cystic stage is very rare. 

Since the head, or scolex, is the ancestor from which 
the worm is formed in the intestine, it is important, 
after giving a vermifuge, to make certain that the head 
has been passed with the worm. Should it remain, a 
new worm will develop. 

The principal tapeworms found in man belong to 
the genera Taenia, H>Tnenolepis, and Dibothriocephalus. 



PHYLUM VERMIDEA 347 

1. Genus Taenia. — (i) Taenia Saginata or T. Medio- 
canellata (Fig. 120). — This, the beef tapeworm, is the 
common tapeworm of the United States. Its length 
sometimes exceeds twenty-five feet. The middle seg- 
ments measure about 6 by 15 mm. The scolex is 
about the size of a pin-head, and is surrounded by four 
sucking discs, but has no booklets (Fig. 122). The uterus 
extends along the midline of the segment and gives 
off about twenty branches upon each side (Fig. 129). 



Fig. 121. — Eggs of Taenia saginata, magnifications loo, 250, and 500 diameters (photo- 
graphs by the author). 

The larval stage is passed in the muscles of various 
animals, especially cattle. 

The scolex is ingested with the meat, its capsule is 
dissolved by the digestive juices, and it attaches itself to 
the intestinal wall by means of its suckers. It then 
develops into the mature worm. 

The ova are present in the stools of infected persons, 
often in great numbers They are spheric or ovoid, 
yellow in color, and have a thick, radially striated shell 
(Fig. 121). Their greatest diameter is 30 to 40 (J^ (about 
four or five times the diameter of a red blood-corpuscle) . 



348 ANIMAL PARASITES 

Vegetable cells, which are generally present in the feces, 
are often mistaken for them. 

(2) Taenia solium, the pork tapeworm, is very rare 
in this country. It is usually much shorter than Tania 
saginata. The scolex is surrounded by four sucking 
discs, and has a projection, or rostellum, with a double 
row of horny booklets (Fig. 123). The uterus has only 
seven to ten branches (Fig. 129). 





Fig. 122. — Head of Taenia saginata (Mos- Fig. 123. — Head of Tsenia solium (Mosler 
ler and Peiper). and Pe-per). 



The cysticercus stage occurs ordinarily in the muscles 
of the pig, but is occasionally seen in man, most fre- 
quently affecting the brain and eye {Cysticercus celluloses). 

The ova closely resemble those of Tcenia saginata, but 
are a little smaller (Fig. 130). 

(3) Taenia Echinococcus. — The mature form of this 
tapeworm inhabits the intestines of the dog and wolf. 
The larvae develop in cattle and sheep ordinarily, but are 
sometimes found in man, where they give rise to echino- 
coccus or " hvdatid" disease. The condition is unusual 



PHYLTJM VEkMIDEA 



349 




in America, but is not infrequent in Central Europe and 
is common in Iceland and Australia. 

The adult parasite is 2.5 to 5 mm. long and consists 
of only four segments (Fig. 124). It contains many ova. 
When the ova reach the digestive tract of man the em- 
bryos are set free and find their way 
to the liver, lung, or other organ, 
where they develop into cysts, thus 
losing their identity. The cysts may 
attain the size of a child's head. 
Other cysts, called " daughter-cysts," 
are formed within these. The cyst- 
wall is made up of two layers, from 
the inner of which develop larvae 
which are identical with the head, or 
scolex, of the mature parasite. These 
are ovoid structures 0.2 to 0.3 mm. 
long. Each has four lateral suckers 
and a rostellum surmounted by a double circular row 
of horny booklets. The rostellum with its booklets is 
frequently invaginated into the body. 

Diagnosis of echinococcus disease depends upon de- 
tection of scolices, free booklets which have fallen off 
from degenerated scolices, or particles of cyst- wall, which 
is characteristically laminated and usually has curled 
edges. The lamination is best seen at the torn edge of 
the membrane. These can be found in fluid withdrawn 
from the cysts or, less frequently, in the sputum or the 
urine, when the disease involves the lung or kidney (Figs. 
•59 and 125). The cysts are sometimes "barren," grow- 
ing to a considerable size without producing scolices. 

The cyst fluid is clear, between 1.009 ^^^ i-oi5 in 



Fig. 124. — Taenia echi- 
nococcus; enlarged (Mos- 
ler and Peiper). 



350 ANIMAL PARASITES 

specific gravity, and contains a notable amount of sodium 
chlorid, but no albumin. 
2. Genus Hymenolepis.— (i) Hymenolepis nana, the 

dwarf tapeworm (Fig. 126), is i to 1.5 cm. in length 
and 0.5 to 0.7 mm. in breadth at the widest part. The 
head is globular and has a rostellum with a crown of 24 







Fig. 125. — Scoles and booklets of Taenia echinococcus in fluid from hepatic cyst (X300) 
(photographs by the author). 

to 30 hooklets. There are about 150 segments The eggs 
are round or oval, 30 to 40 u in diameter, and resemble 
those of TcEuia saginata. The worm is common in Europe 
and America. It is most frequent in children and is gen- 
erally present in large numbers, producing considerable 
digestive and ner\-ous disturbances. The mode of in- 
fection is unknown. 



PHYLUM VERMIDEA r , r- r- ^"^I 

3. Genus Dipylidium.— t^y Dipylidrum r^cT^ipfn^p^,/; 
sometimes called Tania g//i^i?Jci,ill$,fVp;:3f cpiiqan(ion,l^ap^-v- 
worm of dogs and cats. It is about 20 cm. long and 2 to 
3 mm. broad. The intermediate host is the flea or 
louse. Infection of human beings is not common, and 
is mostly confined to children, who are 
probably infected from the dog licking 
their mouths or from getting lice or fleas 
into their mouths. 

4. Genus Dibothriocephalus. — (i) 
Dibothriocephalus latus, the fish tape- 
worm, sometimes reaches fifty feet in 
length, although it is generally not more 
than half so long. When several worms 
are present, they are much smaller. It 
is common in some countries of Europe, especially Ire- 
land, and in Japan, but is very rare in this country, 



nu 




Fig. 126. — Hymen- 
olepis nana, about 
natural size (Mosler 
and Peiper). 




Fig. 127. — Head of Dibothriocephalus latus (about Xg): a, a, Head grooves; 6, neck 

(Blanchard). 

The head is about i mm. broad and is not unlike the 
bowl of a spoon in shape. It is unprovided with either 
suckers or booklets, but has two longitudinal grooves 
which serve the same purpose (Fig. 127) The length of 
the segments is generally less than their breadth, mature 
segments measuring about 3 by 10 or 12 mm. The 
uterus, which is situated in the center of the segment, 
is roset shaped (Fig. 129) and brown or black in color. 
The larval stage is found in fish, especially the pike. 



352 



ANIMAL PARASITES 






• 




Fig. 128. — Ova of Dibothriocephalus latus (X 250 and scx>). The lids were forced open 
by pressure upon the cover-glass (photographs by the author). 




Fig. 129. — Segments of — ft) Tsnia saginata; (2) Dibothriocephalus latus; (3) Taenia 
solium, showing arrangement of uterus. 



The ova are characteristic. They measure about 45 
by 70 ^, are brown in color, and are filled with small 



PHYLUM VERMIDEA 



353 



spherules. The shell is thin and has a small hinged lid 
at one end. As the eggs appear in the feces the Ud is 
not easily seen, but it may be demonstrated by sufl&cient 
pressure upon the cover-glass to force it open (Fig. 1 28) . 
The only other operculated eggs met with in man are 
those of the fluke- worms. 





a b c d e 

Fig. 130. — Comparative size of eggs of intestinal parasites (about X400): a. Taenia 
solium; b. Taenia saginata; c, Ascaris lumbricoides; d, Trichocephalus trichiurus; e, 
Oxyuris vermicularis (after Striimpell). 

Dihothriocephalus latus is interesting clinically because 
it often causes a very severe grade of anemia, which may 
be indistinguishable from pernicious anemia. 



SUBPHYLUM NEMATHELMINTHES 
Qass Nematoda 

The nematodes, or round- worms, are cyhndric or fusi- 
form worms, varying in length, according to species, 
from I mm,' to 40 or 80 cm. As a rule, the sexes are 
separate. The male is smaller and more slender than 
the female. In a few cases the female is viviparous; in 
most cases she deposits ova which are characteristic, 
'so that the finding of a single egg may establish the 
diagnosis. Except in a few instances the young are 
different from the adult, and must pass a certain larval 
stage of development before again reaching a host. 

23 



354 



ANIMAL PARASITES 



An intermediate host is, however, necessary with only 
a few species. 

1. Genus Anguillula. — (i) Anguillula Aceti. — This 

worm, commonly called the "vinegar eel," is usually 
present in vinegar. A drop of the vinegar, particularly 
of the sediment, will frequently show great numbers, all 
in active motion: males, about i or 1.5 mm. long; females, 

somewhat larger and fre- 
quently containing several 
coiled embryos; and young, 
of all sizes up to the adult 
(Fig. 60). 

The vinegar eel is never 
parasitic, but is occasion- 
ally met with as a contami- 
nation in the urine (see p. 
171), and has there been 
mistaken for the larva of 
filaria or strongyloides. 

2. Genus Ascaris. — (i) 
Ascaris Lumbricoides. — 
The female is 20 to 40 cm. 
long and about 6 mm. thick 
(Fig. 131); the male, a little 
more than half as large 
Their color is reddish or 
brown. They are the com- 
mon " round- worms " so 
frequently found in children. Their habitat is the 
small intestine. Large numbers are sometimes present. 
The diagnosis is made by detection of the worms or ova 
in the feces. The latter are generally numerous. They 




Fig. 131. 



-Ascaris lumbricoides (female) 
(Mosler and Peiper). 



PHYLUM VERMIDEA 



355 



are elliptic, measuring about 50 by 70 |U, and have an un- 
segmented protoplasm (Fig. 132). The shell is thick 
and is surrounded by an uneven gelatinous envelop which 
is often stained with bile. 

The eggs do not hatch in the intestine of the original 
host. They pass out in the feces and, after a variable 
period, usually about five weeks, come to contain an 
embryo which remains within the shell until ingested 
by a new host. The embryo is very resistant and may 




Fig. 132. — Ova of Ascaris lumbricoides (X2S0 and 500) (photographs by the author). 



remain alive within the shell for years. Upon reaching 
the intestine of the new host it hatches out and develops 
into the adult worm. 

3. Genus Oxyuris. — (i) Oxyuris Vermicularis. — This 
is the "thread- worm" or "pin-worm" which inhabits 
'the colon and rectum, especially of young children. Its 
presence should be suspected in all unexplained cases of 
pruritus ani. The female is about i cm. long; the male, 
about 0.6 cm. (Fig. 133). 



356 ANIMAL PARASITES 

The worms are not infrequently found in the feces; the 
ova, rarely. The latter are best found by scraping the 
skin at the margin of the anus, where they are deposited 
by the female, who wanders out from the rectum for this 
purpose, this producing the troublesome itching. They 
are asymmetrically oval with one flattened side, are about 
50 u in length, and often contain a partially developed 
embryo. The diagnosis is best made by giving a pur- 
gative and searching the stool for the adult worms. 

Infection takes place through swallowing the ova. 
Auto-infection is likely to occur in children; the ova 
cling to the fingers after scratching and are thus carried 
to the mouth. 




Fig. 133. — Oxyuris vermicularis and egg: a. Male and female, natural size; b, egg (about 
X 250) (after Heller). 



4. Genus Filaria.— (i) Filaria Bancrofti.— The adults 
are thread-like worms, the male about 4 cm., the female 
about 8 cm., long. They live in pairs in the l>Tnphatic 
channels and glands, especially those of the pelvis and 
groin, and often occur in such numbers as to obstruct 
the flow of lymph. This is the most common cause of 
elephantiasis. Infection is very common in tropical 
countries, especially in Samoa, the West Indies, Central 
America, and the Isthmus of Panama. It is said that in 
Samoa 50 per cent, of the natives are infected. 

The female is viviparous, and produces vast numbers 



PHYLUM VERMIDEA 357 

of embryos, which appear in the circulating blood. The 
name Filaria sanguinis hominis, which is commonly 
applied to them, is incorrect, since they do not consti- 
tute a species. These embryos are about as wide 
as a red corpuscle and 0.2 to 0.4 mm. long (Fig. 99), 
and are very actively motile. They are found in 
the peripheral blood only at night, appearing about 8 
P. M., and reaching their maximum number — which is 




Fig. 134. — Embryo of Filaria bancrofti in chylous hydrocele flmd; length, zoo /n; 
width, 8 >i. A number of red blood-corpuscles also appear (studied through courtesy 
of Dr. S. D. Van Meter). 



sometimes enormous — about midnight. If the patient 
change his time of sleeping, they will appear during the 
day. Infection is carried by a variety of mosquito, 
which acts as intermediate host. Diagnosis rests upon 
detection of embryos in the blood, as described on p. 256. 
The embryos are sometimes found in urine and 
chylous fluids from the serous cavities. Their motion is 
then usually less active than when in blood. That shown 
in Fig. 134 was ahve sixty hours after removal of the 



35^ ANIMAL P.\RASITES 

fluid. Embryos were present in the blood of the same 
patient. 

A number of other filariae whose larvse appear in the 
blood are known, some of them only in the larval stage. 
Among these are Filaria philippinensis and F. perstans, 
which exhibit no periodicity, and F. diiirna and F. loa, 
whose embryos appear in the blood during the day. 
The adult of the last named is especially frequent in the 
orbit and beneath the conjunctiva. 

(2) Filaria medinensis, the "guinea-worm," is a very 
interesting and important worm of Africa and southern 
Asia. It is thought to be the " fiery serpent " which 
molested the Children of Israel in the Wilderness. 

The larva probably enters the body through the skin 
or gastro-intestinal tract. It wanders about in the sub- 
cutaneous tissues until maturity, producing slight, if any, 
symptoms. The male has only recently been discovered. 
It is only 4 cm. long. It dies soon after the female is 
impregnated. The adult female is a very slender, 
yellowish worm, about 50 to 80 cm. long, its appearance 
somewhat suggesting a catgut suture. When gestation 
is complete the greater part of the female's body consists 
of a uterus filled with embryos. The female then travels 
to the feet or ankles of the host and there produces a red 
nodule and, finally, an ulcer, from the center of which 
her head protrudes. Through this great numbers of 
embryos are discharged whenever it comes in contact 
with water. Little damage is done unless the worm is 
pulled out, when the embryos are set free in the tissues 
and cause serious disturbances. 

WTien discharged the embryos seek out a small crus- 
tacean, Cyclops, which serves as intermediate host. 



PHYLUM VERMIDEA 



359 



5. Uncinaria Duodenalis and Necator Americanus. 

— These, the Old and the New World hook-worm res- 
pectively, are among the more harmful of the animal 
parasites. They inhabit the small intestine, often in 
great numbers, and commonly produce a severe and often 
fatal anemia. The presence of a few, however, may 
cause slight, if any, disturbance. 




Fig. I3S- — Uncinaria duodenalis: a, Male (natural size); b, female (natural size); c, male 
(enlarged); d, female (enlarged); e, head; /, /, /, eggs (after v. Jaksch). 

Uncinaria duodenalis is common in southern Europe 
and in Egypt. The body is cylindric, reddish in color, 
and the head is bent sharply. The oral cavity has 
six hook-like teeth. The female is 12 to 18 mm. long 
and the tail is pointed. The male is 8 to 10 mm. long and 
the posterior end is expanded into an umbrella-like pouch, 
the caudal bursa. The eggs are oval and have a thin, 
smooth, transparent shell. As they appear in the feces 
the protoplasm is divided into 2, 4, 8, or more rounded 



360 



ANIMAL PARASITES 



segments (Fig. 135). They measure 32 to 40 |t^ by 55 to 

Necator americanus is very common in subtropical 
America, including the southern part of the United 
States and the West Indies. In Porto Rico 90 per cent, 
of the rural population is infected. Isolated cases, prob- 
ably imported, have been seen in most of the Northern 




Fig. 136. — Four eggs of the New World hook-woriu ^iSccator americanus), in the 
one-, two-, and four-cell stages. The egg showing three cells is a lateral view of a four- 
cell stage (about X3S0) (after Stiles). 

States. The American hook-worm is smaller than the 
Old World variety, the male being 7 to 9 mm. long, the 
female 9 to 1 1 mm. The four ventral hook-Hke teeth are 
replaced by chitinous plates. There are also differences 
in the caudal bursa of the male, and in the situation of 
the vulva in the female. The ova (Fig. 136) resemble 
those of Uficinaria duodenalis, but are larger, 36 to 4.0^ 
by 67 to 75 y.. 
The life-history of the two worms is probably the same. 



PHYLUM VERMIDEA 36 1 

The ova pass out with the feces, and, under favorable con- 
ditions of warmth and moisture, develop an embryo 
which hatches within a few days. The resulting larvae 
pass through a stage of development in warm moist 
earth, growing to a length of 0.5 to 0.6 mm., and moulting 
twice. They are then ready to infect a new host. In 
some cases they probably reach the host's intestine by 
way of the mouth, with food or water; but the usual 
route is probably that established by Loos. When moist 
earth containing the larvae comes in contact with the 
skin, they penetrate into the subcutaneous tissues. 
This is favored by retention of mud between the toes of 
those who go barefooted. When the larvae are abundant 
a dermatitis is induced {" ground itch"). From the sub- 
cutaneous tissue they pass by way of lymph- and blood- 
streams to the lungs. Here they make their way into 
the smaller bronchi, are carried by the bronchial mucus 
to the pharynx, and are swallowed. They thus ulti- 
mately reach the small intestine, where they develop 
into mature worms. 

The diagnosis of hook-worm infection, which is assum- 
ing increasing importance in this country, must rest upon 
detection of ova in the ^eces. The worms themselves 
seldom appear except after thymol and a cathartic. A 
small portion of the feces, diluted with water if necessary, 
is placed upon a slide, covered, and searched with a 16 
mm. objective. A higher power may rarely be neces- 
.sary to positively identify an egg, but should not be 
used as a finder. The eggs are nearly always typic, 
showing a thin but very distinct shell, a clear zone, and 
a segmented protoplasm, and after having once been seen 
are not easily mistaken. In severe infections eggs may 



362 ANIMAL PARASITES 

be found in every microscopic field; in most cases, even 
though comparatively mild, they can be found on the first 
slide examined. It is seldom necessary to search more 
than half a dozen slides. When they are scarce, some 
method of sedimenting the feces may be tried, but this 
is rarely necessary. 

6. Genus Strongyloides.— (i) Strongyloides Intes- 
tinalis. — Infection with this worm is by no means so rare 
in this country as the few clinical reports would indicate. 
It is very common in subtropical countries, notably in 
Italy and in southern China. It seems probable that 
the parasite is the cause of " Cochin China diarrhea," 
although some authorities regard it as harmless. 

The adult worm, which reproduces by parthenogenesis, 
is about 2 mm. long. It inhabits the upper portion of 
the small intestine, but neither it nor the ova appear in 
the stool unless an active diarrhea exists. Ordinarily 
the eggs hatch in the intestines, and when infection is 
severe embryos can be found in the feces in large num- 
bers. These are the '' rhabditiform embryos," which 
measure about 0.40 by 0.02 mm. They are actively 
motile, and are best found by making a small depression 
in the fecal mass, filling it with water, and keeping in a 
warm place (preferably an incubator) for twelve to 
twenty-four hours. The embryos will collect in the 
water, and can be easily found by transferring a drop 
to a slide and examining with a 16 mm. objective. The 
inexperienced worker should make sure that the worms 
move, or he may be misled by the vegetable spines 
which are generally present in the feces. ' Certain of 
these spines (notably those from the skin of a peach) 
closely resemble small worms. 



PHYLUM VERMIDEA 363 

Outside the body the rhabditiform embryos develop 
into a free-Hving, sexually differentiated generation. The 
young of this generation are the more slender "filari- 
form embryos" (Fig. 137). Infection can occur either 
through these embryos of the free-living generation or by 
direct transformation of rhabditiform into filariform em- 
bryos, and these into the parthenogenic parasitic adult. 




Fig. 137. — Strongyloides intestinalis: A, Mature female; B, rhabditiform larva; C, filari- 
form larva (after Braun). 

7. Genus Trichinella. — (i) Trichinella Spiralis. — 

This is a very small worm, not exceeding 3 mm. in 
length when fully developed. Infection in man occurs 
from ingestion of insufficiently cooked pork, which 
contains encysted embryos. Ordinary "curing" of 
pork does not kill them. These reach maturity in 
the small intestine. Soon after copulation the males 
die, and the females penetrate into the mucous mem- 
brane. They live in this situation about six weeks, 
giving birth to great numbers of young, averaging as 
high as 1500 from a single female. The larvae migrate 
to the striated muscles, chiefly near the tendinous inser- 
tions, where they grow to a length of about 0.8 mm., and 
finally become encysted. In this condition they may 
remain alive and capable of developing for as long as 
twenty-five years. 



364 ANIMAL PARASITES 

Trichiniasis is generally accompanied by a marked 
eosinophilia. The diagnosis is made by teasing out upon 
a slide a bit of muscle, obtained preferably from the outer 
head of the gastrocnemius, the insertion of the deltoid, 




Fit;. 138. — Trichinella spiralis (larvae) from head of right gastrocnemius muscle; seventh 
week of disease (two-thirds objective; eye-piece 4) (Boston). 

or the lower portion of the biceps. The coiled embryos 
can easily be seen with a i6 mm. objective (Fig. 138). 
The embryos can be found in the blood (p. 257) before 
they have reached their final resting-place in the muscles. 




Fis. 13Q. — Trichocephalus trichiurus: a. Female; b, male (natural size) (Heller). 

8. Genus Trichocephalus.— (i) Trichocephalus Tri- 
chiurus. — This, the "whip-worm," is 4 or 5 cm. long. 
Its anterior portion is slender and thread-like, while 
the posterior portion is thicker (Fig. 139). It is 



PHYLUM VERMIDE 365 

widely distributed geographically, and is one of the most 
common of intestinal parasites in this country. It lives 
in the large intestine, especially the cecum, with its 
slender extremity embedded in the mucous membrane. 
Whip- worms do not, as a rule, produce any symptoms, 
although gastro-intestinal disturbances, nervous symp- 
toms, and anemia have been ascribed to them. They, 
as well as many other intestinal parasites, are probably 




Fig. 140. — Ova of Trichocephalus trichiurus ( X 250 and 500) (photographs by the author). 

an important factor in the etiology of appendicitis, 
typhoid fever, and other intestinal infections. The 
damage which they do to the mucous membrane favors 
bacterial invasion. 

The number present is usually small. The worms 
themselves are rarely found in the feces. The ova, which 
are not often abundant, are easily recognized. They 
are brown, ovoid in shape, about 50 fi long, and have a 
button-like projection at each end (Fig. 140)- 



366 ANIMAL PARASITES 

PHYLUM ARTHROPODA 

The arthropoda which are parasitic to man belong to 
the classes Arachnoidea and Insecta. They are nearly 
all external parasites, and the reader is referred to the 
standard works upon diseases of the skin for descriptions. 
The several species of the louse {Pediculus capitis, P. 
vcslimenti, P. pubis), the itch mite {Sarcoptes scahiei), and 
the small organism {Demodex folliculorum) which lives 
in the sebaceous glands, especially about the face, are 
the most common members of this group. 

A number of flies may deposit their ova in wounds or in 
such of the body cavities as they can reach, and the re- 
sulting maggots may cause intense irritation. Ova may 
be swallowed with the food and the maggots appear in the 
feces. Probably most important is the "screw worm," 
the larva of Chrysomyia macellaria, infection with which 
is not rare in some parts of the United States. The ova 
are most commonly deposited in the nasal passages, and 
the larvae, which may be present in great numbers, 
burrow through the soft parts, cartilage, and even bone, 
always with serious and often with fatal results. 



CHAPTER VII 

MISCELLANEOUS EXAMINATIONS 

PUS 

Pus contains much granular debris and numerous more 
or less degenerated cells, the great majority being poly- 
morphonuclear leukocytes — so-called "pus-corpuscles." 
EosinophiHc leukocytes are common in gonorrheal pus 
and in asthmatic sputum. Examination of pus is di- 
rected chiefly to detection of bacteria. 

When very few bacteria are present, culture methods, 
which are outlined in Chapter VIII, must be resorted to. 
When considerable numbers are present, they can be 
detected and often identified in cover-glass smears. 
Several smears should be made, dried, and fixed, as 
described on p. 407. 

One of these should be stained with a bacterial stain, 
Loffler's methylene-blue and Pappenheim's pyronin- 
methyl-green are especially satisfactory for this pur- 
pose. These stains are applied for one-half minute 
to two minutes or longer, without heating; the prep- 
aration is rinsed in water, dried, mounted, and examined 
with an oil-immersion lens. Another smear should be 
stained by Gram's method. These will give information 
concerning all bacteria which may be present, and fre- 
quently no other procedure will be necessary for their 
identification. 

367 



368 MISCELLANEOUS EXAMINATIONS 

The most common pus-producing organisms are 
staphylococci and streptococci. They are both cocci, or 
spheres, their average diameter being about i ^. Staphy- 
lococci are commonly grouped in clusters, often compared 
to bunches of grapes (Fig. 141). There are several 
varieties which can be distinguished only in cultures. 
Streptococci are arranged side by side, forming chains 
of variable length (Fig. 142). Sometimes there are only 
three or four individuals in a chain; sometimes a chain 




Fig. 141. — Staphylococcus pyogenes albus from an abscess of the parotid gland (Jakob). 

is so long as to extend across several microscopic fields. 
Streptococci are more virulent than staphylococci, and 
are less commonly met. Both are Gram-positive. 
Their cultural characteristics are given on p. 415. 

Should bacteria resembling pneumococci be found, 
Buerger's or Smith's method for capsules (p. 55) should 
be tried. When these are not available, capsules can 
usually be shown by the method of Hiss. The dried 
and fixed smear is covered with a stain composed of 5 c.c. 
saturated alcoholic solution gentian-violet and 95 c.c. 



PUS 369 

distilled water, and heated until steam rises. The prep- 
aration is then washed with 20 per cent, solution of 
copper sulphate, dried, and mounted in Canada balsam. 

Pneumococci may give rise to inflammation in many 
locations (see p. 54). When they form short chains, 
demonstration of the capsule is necessary to distinguish 
them from streptococci. 

If tuberculosis be suspected, the smears should be 
stained by one of the methods for the tubercle bacillus 




Fig. 142. — Streptococcus pyogenes from a case of empyema (Jakob). 

(pp. 49 and 51), or guinea-pigs may be inoculated. 
The bacilli are generally diflicult to find in pus, and 
bacteria-free pus would suggest tuberculosis. 

Gonococci, when typic, can usually be identified with 
sufficient certainty for clinical purposes in the smear 
stained with Loffler's methylene-blue or, much better, 
Pappenheim's pyronin -methyl-green. They are coffee- 
bean-shaped cocci which lie in pairs with their flat sur- 
faces together (Fig. 144). They lie for the most part 
within pus-cells, an occasional cell being filled with them, 

24 



370 



MISCELLANEOUS EXAMINATIONS 



while the surrounding cells contain few or none. A few 
are found outside of the cells. It is not usual to find 
gonococci when many other bacteria are present, even 




Fig. 143. — Diplococcus pneumoniae from ulcer of cornea (obj. one-twelfth oil immersion) 
(study throuKh courtesy of Dr. C. A. Oliver) (Boston). 

though the pus is primarily of gonorrheal origin. When- 
ever the identity of the organism is at all questionable, 
Gram's method should be tried. In rare instances it 




Fig. 144. — Gonococci in urethral pus (McFarland). 

may be necessary to resort to cultures. The gonococcus 
is distinguished by its failure to grow upon ordinary 
media (see p. 416). 



PERITONEAL, PLEURAL, AND PERICARDIAL FLUIDS 37 1 

Gonococci are generally easily found in pus from un- 
treated acute and subacute gonorrheal inflammations — 
conjunctivitis, urethritis, etc. — but are found with diffi- 
culty in pus from chronic inflammations and abscesses, 
and in urinary sediments. 

PERITONEAL, PLEURAL, AND PERICARDLA.L FLUIDS 

The serous cavities contain very little fluid normally, 
but considerable quantities are frequently present as a 
result of pathologic conditions. The pathologic fluids are 
classed as transudates and exudates. 

Transudates are non-inflammatory in origin. They 
contain only a few cells, and less than 2.5 per cent, of 
albumin, and do not coagulate spontaneously. The 
specific gravity is below 1.018. Micro-organisms are 
seldom present. 

Exudates are of inflammatory origin. They are richer 
in cells and albumin, and tend to coagulate upon stand- 
ing. The specific gravity is above 1.018. Bacteria are 
generally present, and often numerous. The amount of 
albumin is estimated by Esbach's method, after diluting 
the fluid. Bacteria are recognized by cultures, animal 
inoculation, or stained smears. 

Exudates are usually classed as serous, serofibrinous, 
seropurulent, purulent, putrid, and hemorrhagic, which 
terms require no explanation. In addition, chylous and 
chyloid exudates are occasionally met, particularly in the 
peritoneal cavity. In the chylous form the milkiness is 
due mainly to the presence of minute fat-droplets, and is 
the result of rupture of a lymph-vessel usually from 
obstruction of the thoracic duct. Chyloid exudates 
are milky chiefly from proteins in suspension, or fine 



372 MISCELLANEOUS EXAMINATIONS 

debris from broken-down cells. These exudates are most 
frequently seen in carcinoma and tuberculosis of the 
peritoneum. 

Cytodiagnosis. — This is diagnosis from a differential 
count of the cells in a transudate or exudate, particularly 
one of pleural or peritoneal origin. 

The fresh fluid, obtained by aspiration, is centrifugal- 
ized for at least five minutes; the supernatant liquid is 




Fig. 145. — Cytodiagnosis. Polymorphonuclear leukocytes and swollen endothelial 
cells from acute infectious non-tuberculous pleuritis (Percy Musgrave; photo by L. S. 
Brown). 

poured off; and cover-glass smears are made and dried in 
the air. The smears are then stained with Wright's 
blood-stain, to which one-third its volume of pure methyl- 
alcohol has been added. Cover the smear with this fluid 
for one-half minute, then dilute with 8 or 10 drops of 
water, and let stand about two minutes. Wash gently 
in water, and dry by holding the cover-glass between the 
fingers over a flame. Mount in balsam and examine 
with an oil-immersion objective. 



PERITONEAL, PLEURAL, AND PERICARDIAL FLUIDS 373 




Fig. 146. — Cytodiagnosis. Lymphoid cells from pleural fluid; case of tuberculous pleuritis 
(Percy Musgrave; photo by L. S. Brown). 




Fig. 147. — Cytodiagnosis. Endothelial cells from transudate or mechanical effusion 
(Percy Musgrave; photo by L. S. Brown). 

Predominance of polymorphonuclear leukocytes (pus- 
corpuscles) points to an acute infectious process (Fig. 

145)- 



374 MISCELLANEOUS EXAMINATIONS 

Predominance of lymphocytes (Fig. 146) generally sig- 
nifies tuberculosis. Tuberculous pleurisy due to direct 
extension from the lung may give excess of polymorpho- 
nuclears owing to mixed infection. 

Predominance of endothelial cells, few cells of any kind 
being present, indicates a transudate (Fig. 147). Endo- 
thelial cells generally predominate in carcinoma, but are 
accompanied by considerable numbers of lymphocytes 
and red blood-corpuscles. 

CEREBROSPINAL FLUID 

Examination of the fluid obtained by lumbar puncture 
is of value in diagnosis of certain forms of meningitis. 

Tubercle bacilli can be found in the majority of cases 
of tuberculous meningitis. The sediment, obtained by 
thorough centrifugahzation or by coagulation and treat- 
ment with antiformin, is spread upon slides and stained 
by one of the methods already given. A consider- 
able number of smears should be examined. In 
doubtful cases inoculation of guinea-pigs must be re- 
sorted to. 

The Diplococcus intracellularis meningitidis is recog- 
nized as the cause of epidemic cerebrospinal fever, and 
can be detected in the cerebrospinal fluid of most cases, 
especially those which run an acute course. Cover-glass 
smears from the sediment should be stained by the 
method for the gonococcus (p. 369). The meningo- 
coccus is an intracellular diplococcus which often cannot 
be distinguished from the gonococcus in stained smears 
(Fig. 148). It, also, decolorizes by Gram's method. The 
presence of such a diplococcus in meningeal exudates is, 
however, sufficient for its identification. 



ANIMAL INOCULATION 375 

Various organisms have been found in other forms of 
meningitis — the pneumococcus most frequently. In 




Fig. 148. — Diplococcus intracellularis meningitidis in leukocytes (X2000) (Wright and 

Brown). 

some cases no micro-organisms can be detected even by 
culture methods. 

ANIMAL INOCULATION 

Inoculation of animals is one of the most reliable means 
of verifying the presence of certain micro-organisms in 
fluids and other pathologic material, and is helpful in 
determining the species of bacteria which have been 
isolated in pure culture. 

Clinically, it is applied almost exclusively to demon- 
stration of the tubercle bacillus when other means have 
failed or are uncertain. The guinea-pig is the most 



376 



MISCELLANEOUS EXAMINATIONS 



suitable animal for this purpose. When the suspected 
material is fluid and contains pus, it should be well cen- 
trifugalized, and one or two cubic centimeters of the 
sediment injected by means of a large hypodermic needle 
into the peritoneal cavity or underneath the loose skin of 
the groin. Fluids from which no sediment can be ob- 
tained must be injected directly into the peritoneal cavity, 
since at least lo c.c. are required, which is too great an 




Fig. i4g. — Influenza bacilli in spinal fluid. Case of meningitis (X looo) (photograph by 

the author). 

amount to inject hypodermically. Sohd material should 
be placed in a pocket made by snipping the skin of the 
groin with scissors, and freeing it from the underlying 
tissues for a short distance around the opening. When 
the intraperitoneal method is selected, several animals 
must be inoculated, since some are likely to die from 
peritonitis caused by other organisms before the tubercle 
bacillus has had time to produce its characteristic lesions. 
The animals should be killed- at the end of six or eight 



THE MOUTH 377 

weeks, if they do not die before that time, and a careful 
postmortem examination should be made for the char- 
acteristic pearl-gray or yellow tubercles scattered over 
the peritoneum and through the abdominal organs, par- 
ticularly the spleen, and for caseous inguinal and retro- 
peritoneal lymph-glands. The tubercles and portions of 
the caseous glands should be crushed between two slides, 
dried, and stained for tubercle bacilli. The bacilli are 
difficult to find in the caseous material. 

THE MOUTH 
Micro-organisms are always present in large numbers. 
Among these is Leptothrix huccalis (Fig. 150), which is 




Fig. 150. — Gingival deposit (iinstained): a. Squamous epithelial cells; b, leujiocs^tes; c, 
bacteria; d, Leptothrix buccalis Qakob). 

especially abundant in the crypts of the tonsils and the 
tartar of the teeth. The whitish patches of Pharyn- 
gomycosis leptothrica are largely composed of these fungi. 
They are slender, segmented threads, which generally, 
but not always, stain violet with Lugol's solution, and are 



378 MISCELLANEOUS EXAMINATIONS 

readily seen with a 4 mm. objective. At times they 
are observed in the sputum and stomach fluid. In the 
former they might be mistaken for elastic fibers; in the 
latter, for Boas-Oppler bacilli. In either case, the re- 
action with iodin will distinguish them. 

Thrush is a disease of the mouth seen most often in 
children, and characterized by the presence of white 
patches upon the mucous membrane. It is caused by the 
thrush fungus, Oidium albicans. When a bit from one 




Fig. 151. — Thrush fungus (Oidium albicans) (Jakob). 

of the patches is pressed out between a slide and cover and 
examined with a 4 mm. objective, the fungus is seen to 
consist of a network of branching segmented hyphae 
with numerous spores, both within the hyphae and in the 
meshes between them (Fig. 151). The meshes also con- 
tain leukocytes, epithelial cells, and granular debris. 

Acute pseudomembranous inflammations, which occur 
chiefly upon the tonsils and nasopharynx, are generally 
caused by the diphtheria bacillus, but may result from 



THE MOUTH 379 

streptococcic infection. In many cases diphtheria 
bacilli can be demonstrated in smears made from the 
membrane and stained with Loffler's methylene-blue or 
2 per cent, aqueous solution of methyl-green. They are 
straight or curved rods, which vary markedly in size 
and outline, and stain very irregularly. A characteristic 
form is a palely tinted rod with several deeply stained 
granules (metachromatic bodies), or with one such 



.. •;■ -^^^ 7^^^-.' j^. 



Fig. 152. — Bacillus diphtheriae stained with methyl-green; culture from throat ( X 1000) 
(photograph by the author). 

granule at each end (Fig. 152). They stain by Gram's 
method. It is generally necessary, and always safer, to 
make a culture upon blood-serum, incubate for twelve 
hours, and examine smears from the growth. 

Vincent's angina is a pseudomembranous and ulcer- 
ative inflammation of mouth and pharynx, which when 
acute may be mistaken for diphtheria, and when chronic 
is very apt to be mistaken for syphilis. Stained smears 



38o 



MISCELLANEOUS EX.\MINATIONS 



from the ulcers or membrane show large numbers of 
spirochaetae and " fusiform bacilli," giving a striking and 
characteristic picture (Fig. 153). The "bacillus" is 
spindle shaped, more or less pointed at the ends, and 
about 6 to 12 a long. The spirillum is a very slender, 
wavy thread, about 30 to 40 u long. Diluted analin- 
gentian-violet makes a satisfactory stain. Further de- 
scription is given on p. 331. 








^. V, 



m ^ 



Fig. 153. — Spirochaeta vincenti from case of ulcerative stomatitis { X 1200) 



Tuberculous ulcerations of mouth and pharynx can 
generally be diagnosed from curetings made after careful 
cleansing of the surface. The curetings are well rubbed 
between slide and cover, and the smears thus made are 
dried, fixed, and stained for tubercle bacilU. Since there 
is much danger of contamination from tuberculous spu- 
tum, the presence of tubercle bacilli is significant only in 
proportion to the thoroughness with which the ulcer was 



THE EYE 381 

cleansed. The diagnosis is certain when the bacilli are 
found within groups of cells which have not been dis- 
associated in making the smears. 

THE EYE 

Staphylococci, pneumococci, and streptococci are prob- 
ably the most common of the bacteria to be found in non- 
specific conjunctivitis and keratitis. Serpiginous ulcer 
of the cornea is generally associated with the pneumococ- 
cus (Fig. 143). 




Fig. 154. — Conjunctival secretion from acute contagious conjunctivitis; polynuclear 
leukocytes with the bacillus of Weeks; P, phagocyte containing bacillus of Weeks (one- 
twelfth oil-immersion, ocular iii) (Morax). 

The usual cause of acute infectious conjunctivitis 
("pink-eye"), especially in cities, seems to be the Koch- 
Weeks bacillus. This is a minute, slender rod, which 
lies within and between the pus-corpuscles (Fig. 154), 
and is negative to Gram's stain. In smears it cannot be 
distinguished from the influenza bacillus, although its 
length is somewhat greater. 

The diplohacillus of Morax and Axenfeld gives rise 
to an acute or chronic blepharoconjunctivitis without 
follicles or membrane, for which zinc sulphate seems to be 
a specific. It is widely distributed geographically and 



382 MISCELLANEOUS EXAMINATIONS 

is common in many regions. The organism is a short, 
thick diplobacillus, is frequently intracellular, and is 
Gram-negative (Fig. 155). A delicate capsule can some- 
times be made out. 

Early diagnosis of gonorrheal ophthalmia is extremely 
important, and can be made with certainty only by detec- 
tion of gonococci in the discharge. They are easily found 
in smears from untreated cases. After treatment is 










Fig. 155. — The diplobacillus of Morax and Axenfeld (from a preparation by Dr. Harold 

Gifford). 

begun they soon disappear, even though the discharge 
continues. 

Pseudomembranous conjunctivitis generally shows 
either streptococci or diphtheria bacilli. In diagnosing 
diphtheritic conjunctivitis, one must be on his guard 
against the Bacillus xerosis, which is a frequent inhabit- 
ant of the conjunctival sac in healthy persons, and which 
is identical morphologically with the diphtheria bacillus. 



THE EAR 383 

The clinical picture is hence more significant than the 
microscopic findings. 

Various micro-organisms — bacteria, molds, protozoa — • 
have been described in connection with trachoma, but the 
specific organism of the disease is not definitely known. 

Herbert has called attention to the abundance of 
eosinophilic leukocytes in the discharge of vernal catarrh. 
He regards their presence in considerable numbers as 
very helpful in the diagnosis of this disease. 

THE EAR 

By far the most frequent exciting causes of acute otitis 
media are the pneumococcus and the streptococcus. The 
finding of other bacteria in the discharge generally indi- 
cates a secondary infection, except in cases complicating 
infectious diseases, such as typhoid fever, diphtheria, and 
influenza. Discharges which have continued for some 
time are practically always contaminated with the 
staphylococcus. The presence of the streptococcus 
should be a cause of uneasiness, since it much more 
frequently leads to mastoid disease and meningitis than 
does the pneumococcus. The staphylococcus, bacillus 
of Friedlander, colon bacillus, and Bacillus pyocyaneus 
may be met in chronic middle-ear disease. 

In tuberculous disease the tubercle bacillus is present in 
the discharge, but its detection offers some difficulties. It 
is rarely easy to find, and precautions must always be 
taken to exclude the smegma and other acid-fast bacilli 
(P- 53)) which are especially liable to be present in the 
ear. Rather striking is the tendency of old squamous 
cells to retain the red stain, and fragments of such cells 
may mislead the unwary. 



384 MISCELLANEOUS EXAMINATIONS 

PARASITIC DISEASES OF THE SKIN 

Favus, tinea versicolor, and the various forms of ring- 
worm are caused by members of the fungus group. To 
demonstrate them, a crust or a hair from the affected area 
is softened with a few drops of 20 per cent, caustic soda 
solution, pressed out between a shde and cover, and 
examined with a one-sixth objective. They consist of a 
more or less dense network of hyphae and numerous 
round or oval refractive spores. The cuts in standard 
works upon diseases of the skin will aid in differentiating 
the members of the group. 

MILK 

A large number of analyses of human and cows' milk 
are averaged by Holt as follows, Jersey milk being ek- 
cluded because of its excessive fat: 

Human Milk. Cows' Milk. 

Normal variations. Average, per Average, per 

per cent. cent. cent. 

Fat 3.00 to 5.CX3 4.00 3.50 

Sugar 6.00 to 7.00 7.CX3 4.30 

Proteins i.cxj to 2.25 1.50 4.00 

Salts 0.18 to 0.25 0.20 0.70 

Water 8Q.82 to 85.50 87. 30 87.50 

100.00 loo.oo 100.00 100.00 

The reaction of human milk is slightly alkaline; of 
cows', neutral or slightly acid. The specific gravity of 
each is about 1.028 to 1.032. Human milk is sterile when 
secreted, but derives a few bacteria from the lacteal 
ducts. Cows' milk, as usually sold, contains large num- 
bers of bacteria, the best milk rarely containing fewer 
than 10,000 per cubic centimeter. Microscopically, 



MILK 



385 



human milk is a fairly homogeneous emulsion of fat, 
and is practically destitute of cellular elements. 

Chemic examination of milk is of great value in solving 
the problems of infant feeding. The sample examined 



r 



\ 




en 



c.c. 

lOl 



2_| 
3_|_ 1 

4_|-6 

6_|-4 
7-|_3 
dJLz 
9_i_ I 



10_=_0 



Fig. 156. — Holt's milk-testing apparatus. 



should be the middle milk, or the entire quantity from 
one breast. The fat and protein can be estimated 
roughly, but accurately enough for many clinical purposes 
by means of Holt's apparatus, which consists of a 10 c.c. 
cream gage and a small hydrometer (Fig. 156). The 

25 



386 



MISCELLANEOUS EXAMINATIONS 



cream gage is filled to the o mark with milk, allowed to 
stand for twenty-four hours at room temperature, and the 
percentage of cream then read off. The percentage of 
fat is three-fifths that of the cream. The protein is then 
approximated from a consideration of the specific gravity 
and the percentage of fat. The salts and sugar very sel- 
dom vary sufficiently to affect the specific gravity, hence 
a high specific gravity must be due to either an increase 
of protein or decrease of fat, or both, and vice versa. 
With normal specific gravity the protein is high when 
the fat is high, and vice versa. The 
method is not accurate with cows' milk. 
For more accurate work the following 
methods, applicable to either human or 
cows' milk, are simple and satisfactory. 
Fat. — Leffmann-Beam Method.- — This 
is essentially the widely used Babcock 
method, modified for the small quanti- 
ties of milk obtainable from the human 
mammary gland. The apparatus con- 
sists of a special tube which fits the 
aluminum shield of the medical centri- 
fuge (Fig. 157) and a 5 c.c. pipet. Owing 
to its narrow stem, the tube is difficult 
to fill and to clean. Exactly 5 c.c. of 
the milk are introduced into the tube by means of the 
pipet, and i c.c. of a mixture of equal parts of concen- 
trated hydrochloric acid and amyl-alcohol is added and 
well mixed. The tube is filled to the o mark with con- 
centrated sulphuric acid, adding a few drops at a time 
and agitating constantly. This is revolved in the centri- 
fuge at 1000 revolutions a minute for three minutes, or 



Fig. iS7.^Tube for 
milk analysis. 



MILK 387 

until the fat has separated. The percentage is then read 
off upon the stem, each small division representing 0.2 
per cent, of fat. 

Proteins. — T. R. Boggs' Modification of the Esbach 
Method. — This is applied as for urinary albumin (p. 105), 
substituting Boggs' reagent for Esbach's. The reagent 
is prepared as follows: 

(i) Phosphotungstic acid 25 gm. 

Distilled water 125 c.c. 

(2) Concentrated hydrochloric acid 25 c.c. 

Distilled water 100 c.c. 

When the phosphotungstic acid is completely dissolved, 
mix the two solutions. This reagent is quite stable if 
kept in a dark glass bottle. 

Before examination, the milk should be diluted accord- 
ing to the probable amount of protein, and allowance 
made in the subsequent reading. For human milk the 
optimum dilution is i : 10; for cows' milk, i : 20. Dilu- 
tion must be accurate. 

Lactose. — The protein should first be removed by 
acidifying with acetic acid, boiling, and filtering. Purdy's 
method may then be used as for glucose in the urine 
(p. 112); but it must be borne in mind that lactose re- 
duces copper more slowly than glucose, and longer heat- 
ing is, therefore, required; and that 35 c.c. of Purdy's 
solution is equivalent to 0.0268 gm. lactose (as com- 
pared with 0.02 gm. glucose). 

It is frequently desirable to detect formalin, which 
is the most common preservative added to cows' milk. 
Add a few drops of dilute ferric chlorid solution to a few 



3^^ 



MISCELLANEOUS EXAMINATIONS 



cubic centimeters of the milk, and run the mixture 
gently upon the surface of some strong sulphuric acid 
in a test-tube. If formaldehyd be present, a bright red 
ring will appear at the line of contact of the fluids. This 
is not a specific test for formaldehyd, but nothing else 
likely to be added to the milk will give it. 

SYPHILITIC MATERIAL 

In 1905 Schaudinn and Hoffmann described the 
occurrence of a very slender, spiral micro-organism in 
the lesions of syphilis. This they named Spirochata 



^ ^^■;^ 




Fig. 158. — Treponema pallidum (X 1000) (Leitz j'j oil-immersion objective and Lcitz 
dark-ground condenser). 

pallida, because of its low refractive power and the 
difficulty with which it takes up staining reagents. The 
name was later changed to Treponema pallidum. Its 
etiologic relation to syphilis is now universally admitted. 
It is found in primary, secondary, and tertiary lesions, 
but is not present in the latter in sufficient numbers to 
be of value in diagnosis. 



SYPHILITIC MATERIAL 389 

Treponema pallidum is an extremely slender, spiral, 
motile thread, with pointed ends. There is a flagellum 
at each end, but it is not seen in ordinary preparations. 
The organism varies considerably in length, the average 
being about 7 /M, or the diameter of a red blood-corpuscle; 
and it exhibits three to twelve, sometimes more, spiral 
curves, which are sharp and regular and resemble the 
curves of a corkscrew (Figs. 112, 158, 159). It is so 
delicate that it is difficult to see even in well-stained 




Fig. 159. — Tr^Mnema pallidum and Spiroch.'eta refringens (X 1200) (Leitz oil-immersion 

objective). 

preparations; a high magnification and careful focusing 
are, therefore, required. Upon ulcerated surfaces it is 
often mingled with other spiral micro-organisms, which 
adds to the difficulty of its detection. The most notable 
of these is Spirochceta refringens, described on p. 333. 

Treponema pallidum is most easily demonstrated in 
chancres and mucous patches, although the skin lesions 
— papules, pustules, roseolous areas— often contain large 
numbers. Tissue-juice from the deeper portions of the 
lesions is the most favorable material for examination, 



390 MISCELLANEOUS EXAMINATIONS 

because the organisms are commonly more abundant 
than upon ulcerated surfaces and are rarely accompanied 
by other micro-organisms. After cleansing, the surface 
is gently scraped with a curet or rubbed briskly with a 
swab of cotton or gauze. In a few moments serum will 
exude. The rubbing should not be so vigorous as to 
bring the blood, because the corpuscles may hide the 
treponema. Very thin cover-glass smears are then made 
from the serum. 

Staining Methods.— Giemsa's stain is probably the most 
widely used. It is best purchased ready prepared. Smears 
are fixed in absolute alcohol for fifteen minutes. Ten drops 
of the stain are added to lo c.c. of faintly alkaline distilled 
water (i drop of a i per cent, solution of potassium carbonate 
to lo c.c. of the water), and the fixed smear is immersed in 
this diluted stain for one to three hours or longer. It is then 
rinsed in distilled water, dried, and mounted. In well-stained 
specimens Treponema pallidum is reddish, most other micro- 
organisms, bluish. More intense staining may be obtained 
by gently warming the stain. 

Wright's blood-stain, used in the manner already described 
(p. 222) except that the diluted stain is allowed to act upon 
the film for fifteen minutes, gives good results. 

Silver Method.— The silver impregnation method has 
long been used for tissues. Stein has applied it to smears as 
follows: 

1. Dry the films in the incubator at 37° C. for three or 
four hours. 

2. Immerse in 10 per cent, silver nitrate solution, in diffuse 
daylight for some hours, until the preparation takes on a 
metallic luster. 

3. Wash in water, dry, and mount. 

The organisms are black against a brownish background. 



SEMEN 391 

India-ink Method. — A drop of India-ink of good grade 
(Gunther and Wagner's recommended) is diluted with i 
to 5 drops of water. A loopful of this is mixed on a slide 
with a similar quantity of serum from the suspected lesion. 
The mixture is then spread over the slide and allowed to dry. 
After drying, it is examined with an oil-immersion lens. 
Micro-organisms, including Treponema pallidum, appear 
clear white on a brown or black background, much as they do 
with the dark ground condenser (Fig. 158). Because of its 
extreme simplicity this method has been favorably received. 
It cannot, however, be absolutely relied upon, since, as has 
been pointed out, many India-inks contain wavy vegetable 
fibrils which might easily mislead a beginner, and sometimes, 
indeed, even an experienced worker. 

Dark groixnd illumination (see p. 21) may be used to 
study the living organisms in fresh tissue juices. This offers 
a satisfactory means of diagnosis, but since the instrument is 
expensive the practitioner will rely upon one or more of the 
staining methods just enumerated. 

SEMEN 

Absence of spermatozoa is a more common cause of 
sterility than is generally recognized. In some cases 
they are present, but lose their motility immediately 
after ejaculation. 

Semen must be kept warm until examined. When it 
must be transported any considerable distance, the 
method suggested by Boston is convenient. The fresh 
semen is placed in a small bottle, to the neck of which a 
string is attached. This is then suspended from a button 
on the trousers, so that the bottle rests against the skin of 
the inguinal region. It may be carried in this way for 
hours. When ready to examine, place a small quantity 



392 



MISCELLANEOUS EXAMINATIONS 



upon a warmed slide and apply a cover. The sperma- 
tozoa are readily seen with a 4 mm. objective (Fig. 57). 
Normally, they are abundant and in active motion. 

Detection of semen in stains upon clothing, etc., is 
often important. The finding of spermatozoa, after 
soaking the stain for an hour in normal salt solution or 




Fig. 160. — Seminal crystals (medium size) (X750) from a stain on clothing. A sin- 
gle thread i inch long was used in the test, the stain being three years and four 
months old (Peterson and Haines). 



dilute alcohol, and teasing in the same fluid, is absolute 
proof that the stain in question is semen, although it is 
not possible to distinguish human semen from that of the 
lower animals in this way. A little eosin added to the 
fluid will bring the spermatozoa out more clearly. 

Florence's Reaction. — The suspected material is soft- 
ened with water, placed upon a slide with a few drops 



DIAGNOSIS OF RABIES 393 

of the reagent, and examined at once with a medium 
power of the microscope. If the material be semen, 
there will be found dark-brown crystals (Fig. i6o) in the 
form of rhombic platelets resembling hemin crystals, or 
of needles, often grouped in clusters. These crystals can 
also be obtained from crushed insects, watery extracts of 
various internal organs, and certain other substances, 
so that they are not absolute proof of the presen'ce of 
semen. Negative results, upon the other hand, are con- 
clusive, even when the semen is many years old. 

The reagent consists of iodin, 2.54 gm.; potassium 
iodid, 1.65 gm.; and distilled water, 30 c.c. 

DIAGNOSIS OF RABIES 

In view of the brilliant results attending prophylactic 
treatment by the Pasteur method, early diagnosis of 
rabies (hydrophobia) in animals which have bitten per- 
sons is extremely important. 

The most reliable means of diagnosis is the production 
of the disease in a rabbit by subdural or intracerebral 
injection of a Httle of the filtrate from an emulsion of 
the brain and medulla of the suspected animal. The 
diagnosis can, however, usually be quickly and easily 
made by microscopic demonstration of Negri bodies. 
Whether these bodies be protozoan in nature and the 
cause of the disease, as is held by many, or whether they 
be products of the disease, it is certain that their presence 
is pathognomonic. 

Negri bodies are sharply outlined, round, oval, or 
somewhat irregular structures which vary in size, the 
extremes being 0.5 and 18 |t/. They consist of a hyalin- 
like cytoplasm, in which when properly stained one or 



394 MISCELLANEOUS EXAMINATIONS 

more chromatin bodies can usually be seen. They are 
situated chiefly within the cytoplasm of the large cells 
of the central nervous system, the favorite locations 
being the multipolar cells of the hippocampus major 
(Ammon's horn). In many cases they suggest red blood- 
corpuscles lying within nerve-cells. 

Probably the best method of demonstrating Negri 
bodies is the impression method of Langdon Frothingham, 
which is carried out as follows: 

(i) Place the dog's brain^ upon a board about lo inches 
square, and divide into two halves by cutting along the me- 
dian line with scissors. 

(2) From one of the halves cut away the cerebellum and 
open the lateral ventricle, exposing the Ammon's horn. 

(3) Dissect out the Ammon's horn as cleanly as possible. 

(4) Cut out a small disc at right angles to the long axis of 
the Ammon's horn, so that it represents a cross-section of the 
organ. 

(5) Place this disc upon the board near the edge, with one 
of the cut surfaces upward. 

(6) Press the surface of a thoroughly clean slide upon the 
disc and lift it suddenly. The disc (if its exposed surface has 
not been allowed to become too dry) will cling to the board, 
leaving only an impression upon the slide. Make several 
similar impressions upon different portions of the slide, using 
somewhat greater pressure each time. Impressions are also 
to be made from the cut surface of the cerebellum, since Negri 
bodies are sometimes present in the Purkinje cells when not 
found in the Ammon's horn. 

(7) Before the impressions dry, immerse in methyl-alcohol 
for one-half to two minutes. 

' For Dr. Frothingham's method of removing a dog's brain .see Ameri- 
can Journal of Public Hygiene for February, 1908. 



PLATE XIII 



V " fi ^J;sr\■^»l-^ 




A , 






.--^' 



% 



Nerve-cells containing Negri bodies. 

Hippocampus impression preparation, dog. Van Gieson stain: 
X looo. I, Negri bodies; 2, capillary; 3, free red blood-corpuscles 
(courtesy of Langdon Frothingham). 



DIAGNOSIS OF RABIES 395 

(8) Cover with Van Gieson's methylene-blue-fuchsin stain, 
warming gently for one-half to two minutes. This stain, as 
modified by Frothingham, is as follows. It must be freshly 
mixed each day: 

Tap-water 20 c.c. 

Saturated alcoholic solution basic fuchsin 3 drops. 

Saturated aqueous solution methylene-blue i drop. 

(9) Wash in water and dry with filter-paper. Examine 
with a low power to locate the large cells in which the 
bodies are apt to be found, and study these with an oil- 
immersion lens. 

The Negri bodies are stained a pale pink to purplish red, 
and frequently contain small blue dots (Plate XIII). The 
nerve-cells are blue, and red blood-corpuscles are colorless 
or yellowish-copper colored. 

When the work is finished, the board with the dissected 
brain is sterilized in the steam sterilizer. 

Demonstration of Negri bodies by this method is quicker 
and, probably, more certain than by the study of sections. 
It has the decided advantage over the smear method that the 
histologic structure is retained. One or more of the impres- 
sions generally shows the entire cell arrangement almost as 
well as in sections, and it is very easy to locate favorable 
fields with a 16 mm. objective. 



CHAPTER VIII 

BACTERIOLOGIC METHODS 

Bacteriology has become so important a part of medi- 
cine that some knowledge of bacteriologic methods is 
imperative for the present-day practitioner. It has been 
the plan of this book to describe the various bacteria 
and bacteriologic methods with the subjects to which 
they seemed to be particularly related. The tubercle 
bacillus and its detection, for example, are described in 
the chapters upon Sputum and Urine; blood-cultures 
are discussed in the chapter upon Blood. There are, 
however, certain methods, notably the preparation of 
media and the study of bacteria by cultures, which do 
not come within the scope of any previous section, and 
an outline of these is given in the present chapter, 

L APPARATUS 

Much of the apparatus of the clinical laboratory is 
called into use. Only the following need special mention : 

I. Sterilizers. — Two are required. 

The dry, or hot-air sterilizer, is a double-walled oven 
similar to the detached ovens used with gas and gasolene 
stoves. It has a hole in the top for a perforated cork 
with thermometer. 

The steam sterilizer is preferably of the Arnold type, 
opening either at the top or the side. An autoclave, which 

39(3 



APPARATUS 397 

sterilizes with steam under pressure, is very desirable, 
but not necessary. 

2. Incubator. — This is the most expensive piece of 
apparatus which will be needed. It is made of copper, 
and has usually both a water- and an air-jacket surround- 
ing the incubating chamber. It is provided with ther- 
mometer, thermo-regulator, and some source of heat, 
usually a Koch safety Bunsen burner. With a little 
ingenuity one can rig up a drawer or a small box, in 
which a fairly constant temperature can be maintained 
by means of an electric light. The degree of heat can 
be regulated by moving the drawer in or out, or holes 
can be made in which corks may be inserted and removed 
as needed. A Thermos bottle has been suggested as a 
temporary make-shift. 

3. Culture-tubes and Flasks. — For most work ordinary 
test-tubes, 5 by | inches, are satisfactory. For special 
purposes a few 3 by | inch and 6 by f inch tubes may 
be needed. Heavy tubes, which do not easily break, can 
be obtained, and are especially desirable when tubes are 
cleaned by an untrained assistant. 

Flasks of various sizes are needed. The Ehrlen- 
meyer type is best. Quart and pint milk bottles and 
2-ounce wide-mouthed bottles will answer for most pur- 
poses. 

4. Platinum Wires. — At least two of these are needed. 
Each consists of a piece of platinum wire about 8 cm. long, 
fixed in the end of a glass or metal rod. One is made of 
about 22 gage wire and its end is curled into a loop i to 
2 mm. in diameter. A loop i mm. in diameter is some- 
times called a " normal." The other wire is somewhat 
heavier and its tip is hammered flat. 



398 BACTERIOLOGIC METHODS 

5. Pipets, etc. — In addition to the graduated pipets 
with which every laboratory is suppHed, there are a 
number of forms which are generally made from glass 
tubing as needed. One of the simplest of these is made 
as follows: A section of glass tubing, about 12 cm. long 
and 8 mm. in diameter, is grasped at the ends, and its 
center is heated in a concentrated flame. A blast-lamp 
is best, but a Bunsen burner will usually answer, par- 



GPOUP> A 



Gr?OUP> B 



Fig. 161. — Process of making pipets (group A) and Wright's capsule (group B). The 
dotted lines indicate where the glass is to be broken. 

ticularly if fitted with a "wing" attachment. When the 
glass is thoroughly softened it is removed from the flame, 
and, with a steady, but not rapid pull, is drawn out as 
shown in Fig. 161. The slender portion is scratched 
near the middle with a file and is broken to make two 
pipets, which are then fitted with rubber nipples. Two 
conditions are essential to success: the glass must be 
thoroughly softened and it must be removed from the 
flame before beginning to pull. 



STERILIZATION 399 

A nipple can be made of a short piece of rubber 
tubing, one end of which is plugged with a glass bead. 

This pipet has many uses about the laboratory. 
When first made it is sterile and may be used to transfer 
cultures. With a grease-pencil mark about 2 cm. from 
its tip (Fig. 163), it is useful for measuring very small 
quantities of fluid, as in making dilutions for the Widal 
test and in counting bacteria in vaccines. Mett's tubes 
for pepsin estimation may be made from the capillary 
portion. The capillary portion also makes a very satis- 
factory blood-lancet if heated in a low flame and drawn 
out quickly. 

Another useful device is the Wright capsule, which 

is made as shown in Fig. 161. Its use is illustrated in 

Fig. ICO. After the straight end is sealed the curved 

portion may be hooked over the aluminum tube of the 

centrifuge, and the contained blood or other fluid sedi- 

mented ; but the speed should not be so great as to break 

the capsule. 

II. STERILIZATION 

All apparatus and materials used in bacteriologic 
work must be sterilized before use. 

Glassware, metal, etc., are heated in the hot-air steril- 
izer at 150° to 180° C. for half an hour. Flasks, bottles, 
and tubes are plugged with cotton before heating. 
Petri dishes may be wrapped in paper in sets of three. 
Pipets and glass and metal h)^odermic syringes are 
placed in cotton-stoppered test-tubes. 

Culture-media and other fluids must be sterilized by 
steam. Exposure in an autoclave to a temperature of 
110° C. (6 pounds pressure) for one-half hour is sufficient. 
With the Arnold sterilizer, which is more commonly 



4CX) BACTERIOLOGIC METHODS 

used, the intermittent plan must be adopted, since 
steam at ordinary pressure will not kill spores. This con- 
sists in steaming for thirty to forty-five minutes on three 
or four successive days. Spores which are not destroyed 
upon the first day develop into the vegetative form and 
are destroyed at the next heating. Gelatin media must 
not be exposed to steam for more than twenty minutes 
at a time, and must then be removed from the sterilizer 
and cooled in cold water, otherwise the gelatin may lose 
its power to solidify. 

Cotton and gauze are sterilized by either hot air or 
steam, preferably the latter. 

III. PREPARATION OF CULTURE-TUBES 

New tubes should be washed in a very dilute solution 
of nitric acid, rinsed in clear water, and allowed to drain 
dry. 

Tubes which contain dried culture-media are cleaned 
with a test-tube brush after boiling in a strong solution of 
washing-soda. They are then rinsed successively in 
clear water, acidulated water, and clear water, and al- 
lowed to drain. 

The tubes are now ready to be plugged with raw cotton 
— the "cotton batting" of the dry goods stores. This is 
done by pushing a wad of cotton into each tube to a 
depth of about 3 cm. with a glass rod. The plugs should 
fit snugly, but not too tightly, and should project from 
the tube sufficiently to be readily grasped by the fingers. 
The tubes are next placed in wire baskets and heated in 
an oven for about one-half hour at 150° C. in order to 
mold the stoppers to the shape of the tubes. The heat- 
ing should not char the cotton, although a sHght brown- 



CULTURE-MEDIA r n r~ 40I 

ing does no harm. The tub^s are noW^sta-i^Y ^f^9 filled 
with culture-media. '' '^-((^(Al,(r r, '^^, ^^- ' // fC 

IV. CULTURE-MEDIA 

For a careful study of bacteria a great variety of culture- 
media is required, but only a few — bouillon, agar or solid- 
ified blood-serum, and gelatin — are much used in routine 
work. 

Preparation of Culture-media. — 

Beef Infusion 

Hamburger steak, lean 500 grams. 

Tap-water icxdo c.c. 

Mix well; let soak about twenty-four hours in an ice- 
chest, and squeeze through cheese-cloth. This infusion 
is not used by itself, but forms the basis for various 
media. *' Double strength " infusion, used in making 
agar-agar, requires equal parts of the meat and water. 

Infusion Bouillon 

Beef infusion 1000 c.c. 

Peptone (Witte) 10 grams. 

Salt 5 " 

Boil until dissolved; bring to original bulk with 
water; adjust reaction; and filter. 

Beef Extract Bouillon 

Liebig's extract of beef 3 grams. 

Peptone 10 " 

Salt 5 " 

Tap-water 1000 c.c. 

When all ingredients are dissolved, cool, and beat 
in the whites of two eggs; boil briskly for five minutes 
and filter. It is not usually necessary to adjust the re- 
action. 

26 



402 ^CTERIOLOGIC METHODS 

■ , ■ o A*-'' " ' Agak-agar 

Preparation of this medium usually gives the student 
much trouble. There should be no difficulty if the direc- 
tions are carefully carried out. 

Agar-agar, powdered or in shreds 15 grams. 

Tap- water • 500 c.c. 

Boil until thoroughly dissolved and add — 

Peptone 10 grams 

Salt S " 

When these have dissolved, replace the water lost in 
boiling, cool to about 60° C, and add 500 c.c. double- 
strength beef infusion. Bring slowly to the boil, adjust- 
ing the reaction meanwhile, and boil for at least five 
minutes. Filter while hot through a moderately thick 
layer of absorbent cotton wet with hot water in a hot 
funnel. A piece of coarse wire gauze should be placed 
in the funnel underneath the cotton to give a larger filter- 
ing surface. This medium will be clear enough for or- 
dinary work. If an especially clear agar is desired, it 
can be filtered through paper in an Arnold sterilizer. 

Agar can also be made by boiling 15 grams of powdered 
agar in 1000 c.c. of bouillon until dissolved, replacing 
the water lost in boiling, and filtering through paper 
in a sterilizer. It can be cleared with egg if desired. 

Glycerin Agar-agar 
To 1000 c.c. melted agar add 60 to 70 c.c. glycerin. 

Gelatin 
Dissolve 100 to 120 grams "golden seal" gelatin in 
1000 c.c. nutrient bouillon with as little heat as possible, 



CULTURE-MEDIA 403 

adjust the reaction, cool, beat in the whites of two eggs, 
boil, and filter hot through filter-paper wet with hot 
water. Sterilize in an Arnold sterilizer for twenty min- 
utes upon three successive days and cool in cold water 
after each heating. 

Sugar Media 
Any desired sugar may be added to bouillon, agar, or 
gelatin in proportion of 10 grams to the liter. Dextrose 
is most frequently required. When other sugars are 
added, media made from beef-extract should be used, 
since those made from beef-infusion contain enough dex- 
trose to cause confusion. 

Loffler's Blood-serum 

I per cent, dextrose-bouillon i part. 

Blood-serum 3 parts. 

Mix and tube. Place in an inspissator at the proper 
slant for three to six hours at 80° to 90° C. When firmly 
coagulated, steriUze in the usual way, A large "double- 
cooker" makes a satisfactory inspissator. The tubes 
are placed in the inner compartment at the proper slant, 
a lid with perforation for a thermometer is apphed, and 
the whole is weighted down in the water of the outer 
compartment. 

Blood-serum is obtained as follows: Beef or pig blood 
is collected in a bucket at the slaughter-house and 
placed in an ice-chest until coagulated. The clot is then 
gently loosened from the wall of the vessel. After about 
twenty-four hours the serum will have separated nicely 
and can be siphoned off. It is then stored in bottles 
with a little chloroform until needed. Red cells, if 
abundant, darken the medium, but do no harm. 



404 BACTERIOLOGIC METHODS 

Solidified blood-serum is probably the most satisfac- 
tory medium for general purposes. Nearly all patho- 
genic organisms grow well upon it. 

Hemoglobin Medium 

The simplest way to prepare this is to smear a drop 
of blood, obtained by puncture of the finger, over the 
surface of an agar-slant, and to incubate over night to 
make sure of sterility. It is used for growing the influ- 
enza bacillus. 

Litmus Milk 

Fresh milk is steamed in an Arnold sterilizer for half 
an hour, and placed in the ice-chest over night. The 
milk is siphoned off from beneath the cream, and sufl&- 
cient aqueous solution of litmus or, preferably, azolitmin 
• is added to give a blue- violet color. It is then tubed 
and sterilized. 

Potato 

Cylinders about one-half inch thick are cut from 
potato and spHt obHquely. These are soaked over 
night in running water and placed in large tubes, in the 
bottom of which is placed a little cotton saturated with 
water. They are sterilized for somewhat longer periods 
than ordinary media. 

Dunham's Peptone Solution 

Peptone lo grams. 

Salt 5 " 

Water looo c.c. 

Dissolve by boiling; filter, "tube, and sterilize. 
This medium is used to determine indol production. 
To a twenty-four- to forty-eight-hour-old culture is 



CULTURE-MEDIA 405 

added 5 to 10 drops of concentrated c. p. sulphuric acid 
and I c.c. of i : 10,000 solution of sodium nitrite. Ap- 
pearance of a pink color shows the presence of indol. 
A pink color before the nitrite is added shows the presence 
of both indol and nitrites. 

Hiss' Serum Media 

Blood-serum i part. 

Water 3 parts. 

Warm and adjust reaction to + 0.2 to + 0.8. Add 
Utmus or azolitmin solution to give a blue-violet color. 
Finally, add i per cent, of inulin or any desired sugar. 
The inulin medium is very useful in distinguishing be- 
tween the pneumococcus and streptococcus. 

Bile Medium 

Ox- or pig-bile is obtained at the slaughter-house, 
tubed, and sterilized. This is used especially for grow- 
ing typhoid bacilli from the blood during Ufe. The fol- 
lowing is probably as satisfactory as fresh bile and is 
more convenient: 

Inspissated ox-bile (Merck) 30.0 grams. 

Peptone 2.5 

Water 250.0 c.c. 

Dissolve, place in tubes, and sterilize. 

Reaction of Media. — The chemic reaction of the 
medium exerts a marked influence upon the growth of 
bacteria. It is adjusted after all ingredients are dis- 
solved by adding sufficient caustic soda solution to 
overcome the acidity of the meat and other substances 
used. In general, the most favorable reaction lies 
between the neutral points of Utmus and phenolphtha- 



4o6 BACTERIOLOGIC METHODS 

lein, representing a very faint alkalinity to litmus. 
In routine work it is usually sufficient to test with 
litmus-paper. When greater accuracy is demanded, 
the following method should be used: After all ingre- 
dients are dissolved and the loss during boiling has 
been replaced with water, lo c.c. of the medium are 
transferred to an evaporating dish, diluted with 40 c.c. 
of water, and boiled for three minutes to drive off carbon 
dioxid. One c.c. of 0.5 per cent. alcohoUc solution of 
phenolphthalein is then added, and decinormal sodium 
hydroxid solution is run in from a buret until the neutral 
point is reached, indicated by the appearance of a per- 
manent pink color. The number of cubic centimeters of 
decinormal solution required to bring this color indicates 
the number of cubic centimeters of normal sodium hy- 
droxid solution which will be required to neutralize 100 
c.c. of the medium . The standard reaction is -I- 1 .5, which 
means that the medium must be of such degree of acidity 
that 1.5 c.c. of normal solution would be required to 
neutralize 100 c.c. This corresponds to faint alkalinity 
to litmus. Most pathogenic bacteria grow better with a 
reaction of +1.0 or -I- 0.8. Example: If the 10 c.c. which 
were titrated required 2 c.c. of decinormal solution to 
bring the pink color, the reaction is +2; and 0.5 c.c. of 
normal sodium hydroxid must be added to each 100 c.c. 
of the medium to reduce it to the standard, +1.5. 

Tubing Culture-media. — The finished product is 
stored in flasks or distributed into test-tubes. This is 
done by means of a funnel fitted with a section of rubber 
tubing with a glass tip and a pinch-cock. Great care 
must be exercised, particularly with media which solid- 
ify, not to smear any of it upon the inside of the mouth of 



STAINING METHODS 407 

the tube, otherwise the cotton stopper will stick. Tubes 
are generally filled to a depth of 3 or 4 cm. For stab- 
cultures a greater depth is desired. 

After tubing, all culture-media must be sterilized as 
already described. Agar-tubes are cooled in a slanting 
position to secure the proper surface for inoculation. 

Media should be stored in a cool place, preferably an 
ice-chest. Evaporation may be prevented by covering 
the tops of the tubes with tin-foil or the rubber caps 
which are sold for the purpose; or the cotton stopper 
may be pushed in a short distance and a cork inserted. 

V. STAINING METHODS 

In general, bacteria are stained to determine their 
morphology, their reaction with special methods (e. g., 
Gram's method), and the presence or absence of certain 
structures, as spores, flagella, and capsules. Staining 
methods for various purposes and the formulae of the 
staining fluids have been given in previous chapters and 
can be found by consulting the index. The following 
will probably be most frequently used: 

Methods for tubercle bacilli, pp. 49, 51, and 168. 

Methods for capsules of bacteria, pp. 55 and 368. 

Methods for Treponema pallidum, p. 390. 

Loffler's alkaline methylene-blue, p. 57. 

Blood-stains, pp. 221-224. 

- The method of staining bacteria for morphology is as 
follows, using any simple bacterial stain: 

(i) Make a thin smear upon a slide or cover-glass. 

(2) Dry in the air, or by warming high above the flame. 



4o8 BACTERIOLOGIC METHODS 

(3) " Fix " bypassing the preparation, film side up, rather 
slowly through the flame of a Bunsen burner: a cover-glass 
three times, a slide about twelve times. Take care not to 
scorch. 

(4) Apply the stain for the necessary length of time, gen- 
erally one-quarter to one minute. 

(5) Wash in water. 

(6) Dry by waving high above a flame or by blotting with 
filter-paper. 

(7) Mount by pressing the cover, film side down, upon a 
drop of Canada balsam on a slide. Slides may be examined 
with the oil-immersion lens without a cover-glass. 

Simple Bacterial Stains. — A simple solution of any 
basic anilin dye (methylene-blue, basic fuchsin, gentian 
violet, etc.) will stain nearly all bacteria. These 
simple solutions are not much used in the clinical 
laboratory, because other stains, such as Loffler's 
methylene-blue and Pappenheim's pyronin-methyl- 
green stain, which serve the purpose even better, are 
at hand. 

Pappenheim's Pyronin-methyl-green Stain. — This so- 
lution colors bacteria red and nuclei of cells blue. It 
is, therefore, especially useful for intracellular bacteria 
like the gonococcus and the influenza bacillus. It is a 
good stain for routine purposes, and is a most excellent 
contrast stain for Gram's method. It colors the cyto- 
plasm of lymphocytes bright red, and has been used as a 
differential stain for these cells. The solution is applied 
cold for one-half to five minutes. It consists of saturated 
aqueous solution methyl-green, 3 to 4 parts, and saturated 
aqueous solution pyronin, i to i| parts. 

Carbol Thionin. — Saturated solution thionin in 50 per 



STAINING METHODS 409 

cent, alcohol, 20 c.c; 2 per cent, aqueous solution phenol, 
icx) c.c. 

This is especially useful in counting bacteria for a 
vaccine (p. 422). It can be used as a general stain. In 
blood work it is used for the malarial parasite and 
for demonstration of basophilic degeneration of the 
red cells. It should be preceded by fixation for about 
a minute in saturated aqueous solution of mercuric 
chlorid. 

Gram's Method. — This is a very useful aid in differen- 
tiating certain bacteria and should be frequently resorted 
to. It depends upon the fact that when treated succes- 
sively with gentian-violet and iodin, certain bacteria 
(owing to formation of insoluble compounds) retain the 
stain when treated with alcohol, whereas others quickly 
lose it. The former are called Gram-positive, the latter, 
Gram-negative. In order to render Gram-negative or- 
ganisms visible, some contrasting counter stain is com- 
monly applied, but this is not a part of Gram's method 
proper. 

(i) Make smears, dry, and fix by heat. 

(2) Apply anilin-gentian-violet or formalin-gentian-violet 
(p. 57) two to five minutes. 

(3) Wash with water. 

(4) Apply Gram's iodin solution one-half to two minutes. 

(5) Wash in alcohol until the purple color ceases to come 
off. This is conveniently done in a watch-glass. The prep- 
aration is placed in the alcohol, face downward, and one 
edge is raised and lowered with a needle. As long as any 
color is coming off, purple streaks will be seen diffusing into 
the alcohol where the surface of the fluid meets the smear. 
If forceps be used, beware of stain which may have dried 



4IO BACTERIOLOGIC METHODS 

upon them. The thinner portions of smears from pus should 
be practically colorless at this stage. 

(6) Apply a contrast stain for one-half to one minute. 
The stains commonly used for this purpose are an aqueous 
or alcoholic solution of Bismarck brown and a weak solution 
of fuchsin. In the writer's experience, Pappenheim's pyro- 
nin-methyl-green mixture is much more satisfactory; it 
brings out Gram-negative bacteria more sharply, and is 
especially desirable for intracellular Gram-negative organ- 
isms like the gonococcus and influenza bacillus, since the 
bacteria are bright red and nuclei of cells blue. 

(7) Wash in water, dry, and mount in balsam. 

The more important bacteria react to this staining 
method as follows: 

Gram Staining Gram Decolorizing 

(Deep purple). (Colorless unless a counterstain is used). 

Staphylococcus. Gonococcus. 

Streptococcus. Meningococcus. 

Pneumococcus. Micrococcus catarrhalis. 

Bacillus diphtheriae. Bacillus of influenza. 

Bacillus tuberculosis. Typhoid bacillus. 

Bacillus of anthrax. Bacillus coli communis. 

Bacillus of tetanus. Spirillum of Asiatic cholera. 

Bacillus aerogenes capsulatus. Bacillus pyocyaneus. 

Bacillus of Friedlander. 

Koch-Weeks bacillus. 

Bacillus of Morax-Axenfeld. 

Moeller's Method for Spores. — Bodies of bacteria are 
blue, spores are red. 

(i) Make thin smears, dry, and fix. 

(2) Wash in chloroform for two minutes. 

(3) Wash in water. 

(4) Apply 5 per cent, solution of chromic acid one-half to 
two minutes. 



STAINING METHODS 4II 

(5) Wash in water. 

(6) Apply carbol-fuchsin and heat to boiling. 

(7) Decolorize in 5 per cent, solution of sulphuric acid. 

(8) Wash in water. 

(9) Apply I per cent, aqueous solution of methylene-blue 
one-half minute. 

(10) Wash in water, dry, and mount. 

Loffler's Method for Flagella. — The methods for 
flagella are applicable only to cultures. Enough of the 
growth from an agar-culture (which should not be more 
than eighteen to twenty-four hours old) to produce faint 
cloudiness is added to distilled water. A small drop of 
this is placed on a cover-glass, spread by tilting, and dried 
quickly. The covers must be absolutely free from grease. 
To insure this, they may be warmed in concentrated 
sulphuric acid, washed in water, and kept in a mixture 
of alcohol and strong ammonia. When used they are 
dried upon a fat-free cloth. 

(i) Fix by heating the cover over a flame while holding 
in the fingers. 

(2) Cover with freshly filtered mordant and gently warm 
for about a minute. 

The mordant consists of: 

20 per cent, aqueous solution of tannic acid 10 c.c. 

Saturated solution ferrous sulphate, cold 5 c.c. 

Saturated aqueous or alcoholic solution gentian violet . . i c.c. 

(3) Wash in water. 

(4) Apply freshly filtered anilin-gentian-violet, warming 
gently for one-half to one minute. 

(5) Wash in water, dry, and mount in balsam. 



412 BACTERIOLOGIC METHODS 

VI. METHODS OF STUDYING BACTERIA 

The purpose of bacteriologic examinations is to de- 
termine whether bacteria are present or not, and, if 
present, their species and comparative numbers. In 
general, this is accomplished by: i, Direct microscopic 
examination; 2, Cultural methods; 3, Animal inocu- 
lation. 

1. Microscopic Examination. — Every bacteriologic 
examination should begin with a microscopic study of 
smears from the pathologic material, stained with a gen- 
eral stain, by Gram's method, and often by the method 
for the tubercle bacillus. This yields a great deal of 
information to the experienced w^orker, and in many cases 
is all that is necessar}- for the purpose in view. It will 
at least give a general idea of what is to be expected, and 
may determine future procedure. 

2. Cultural Methods.— (i) Collection of Material. — 
Material for examination must be collected under abso- 
lutely aseptic conditions. It may be obtained with a 
platinum wire — which has been heated to redness just 
previously and allowed to cool — or with a swab of sterile 
cotton on a stifif wire or wooden applicator. Such swabs 
may be placed in cotton-stoppered test-bubes, sterilized, 
and kept on hand ready for use. Fluids which contain 
very few bacteria, and hence require that a consider- 
able quantity be used, may be collected in a sterile 
hypodermic syringe or one of the pipets described on 
p. 398. The method of obtaining blood for cultures 
is given on p. 245. 

(2) Inoculating Media. — The material is thoroughly 
distributed over the surface of some solid medium, solid- 



METHODS or STUDYING BACTERIA 413 

ified blood-serum being probably the best for routine work. 
When previous examination of smears has shown that 
many bacteria are to be expected, a second tube should 
be inoculated from the first, and a third from the second, 
so as to obtain isolated colonies in at least one of the tubes. 
The platinum wire must be heated to redness before and 
after each inoculation. When only a few organisms of 
a single species are expected, as is the case in blood-cul- 
tures, a considerable quantity of the material is mixed 
with a fluid medium. 

(3) Incubation. — Cultures are placed in an incubator 
which maintains a uniform temperature, usually of 37.5° 
C, for eighteen to twenty-four hours, and the growth, if 
any, is studied as described later. Gelatin will melt with 
this degree of heat, and must be incubated at about 
room-temperature. 

(4) Study of Cultures. — ^When the original culture 
contains more than one species, they must be separated, 
or obtained in "pure culture," before they can be studied 
satisfactorily. To accompHsh this it is necessary to so 
distribute them on solid media that they form separate 
colonies, and to inoculate fresh tubes from the individual 
colonies. In routine work the organisms can be suffi- 
ciently distributed by drawing the infected wire over the 
surface of the medium in a series of streaks. If a suffi- 
cient number of streaks be made, some of them are 
sure to show isolated colonies. Another method of 
obtaining isolated colonies is to inoculate the water 
of condensation of a series of tubes, the first from 
the second, the second from the third, etc., and, by 
tilting, to flow the water once over the surface of the 
medium. 



414 BACTERIOLOGIC METHODS 

In order to determine the species to which an organism 
belongs it is necessary to consider some or all of the fol- 
lowing points: 

(i) Naked-eye and microscopic appearance of the col- 
onies on various media. 

(2) Comparative luxuriance of growth upon various 
media. The influenza bacillus, for example, can be 
grown upon media containing hemoglobin, but not on 
the ordinary media. 

(3) Morphology, special staining reactions, and the 
presence or absence of spores, flagella, and capsules. 
Staining methods for these purposes have been given. 

(4) Motility. This is determined by observing the 
living organism with an oil-immersion lens in a hanging- 
drop preparation, made as follows: A small drop of a 
bouillon culture or of water of condensation from an 
agar or blood-serum tube is placed upon the center of 
a cover-glass; this is inverted over the concavity of a 
" hollow slide," and ringed with vaselin. In focusing, 
the edge of the drop should be brought into the field. 
Great care must be exercised not to break the cover by 
pushing the objective against it. A method which in 
some respects is preferable to the hanging drop is to 
make a ring of vaselin on a slide, place a drop of the 
culture in this, and apply a cover. 

It is not always easy to determine whether an organism 
is or is not motile, since the motion of currents and the 
Brownian motion which affects all particles in suspen- 
sion are sometimes very deceptive. 

(5) Production of chemic changes in the media. 
Among these are coagulation of milk; production of acid 
in milk and various sugar media to which litmus has been 



CHARACTERISTICS OF SPECIAL BACTERIA 415 

added to detect the change; production of gas in sugar 
media, the bacteria being grown in fermentation tubes 
similar to those used for sugar tests in urine; and pro- 
duction of indol. 

(6) Ability to grow without oxygen. For anaerobic 
methods, the reader is referred to larger works. 

(7) Effects produced when inoculated into animals. 
3. Animal Inoculation. — In clinical work this is 

resorted to chiefly to detect the tubercle bacillus. The 
method is described on p. 375. 

For the study of bacteria in cultures, a small amount 
of a pure culture is injected subcutaneously or into the 
peritoneal cavity. The animals most used are guinea- 
pigs, rabbits, and mice. For intravenous injection the 
rabbit is used because of the easily accessible marginal 
vein of the ear. 

VII. CHARACTERISTICS OF SPECIAL BACTERIA 

Owing to the great number of bacterial species, most 
of which have not been adequately studied, positive 
identification of an unknown organism is often a very 
difficult problem. Fortunately, however, only a few 
are commonly encountered in routine work, and identi- 
fication of these with comparative certainty presents no 
great difficulty. Their more distinctive characteristics 
are outlined in this section. 

I . Staphylococcus Pyogenes Aureus.— The morphology 
and staining reactions (described on p. 368) and the ap- 
pearance of the colonies are sufficient for diagnosis. 
Colonies on solidified blood-serum and agar are rounded, 
slightly elevated, smooth and shining, and vary in color 



4l6 BACTERIOLOGIC METHODS 

from light yellow to deep orange. Young colonies are 
sometimes white. 

2. Staphylococcus Pyogenes Albus. — This is similar 
to the above, but colonies are white. It is generally 
less virulent. 

3. Staphylococcus Pyogenes Citreus. — The colonies 
are lemon yellow; otherwise it resembles the white 
staphylococcus. 

4. Streptococcus F*yogenes. — The morphology and 
staining reactions have been described (p. 368). The 
chains are best seen in the water of condensation and in 
bouillon cultures. The cocci are not motile. Colonies 
on blood-serum are minute, round, grayish, and trans- 
lucent. Litmus milk is usually acidified and coagulated, 
although slowly. The streptococcus rarely produces 
acid in Hiss' serum-water-litmus-inulin medium (see 
p. 405). 

5. Pneumococcus. — The only organism with which this 
is likely to be confused is the streptococcus. The dis- 
tinction is often extremely difficult. 

Detection of the pneumococcus in fresh material has 
been described (p. 54). In cultures it frequently forms 
long chains. Capsules are not present in cultures ex- 
cept upon special media. They show best upon a 
serum medium like that described for the gonococcus, 
but can frequently be seen in milk. Colonies on blood- 
serum resemble those of the streptococcus. The pneumo- 
coccus usually promptly acidifies and coagulates milk, 
and acidifies and coagulates Hiss' serum-water with 
inulin. The latter property is very helpful in diag- 
nosis. 

6. Gonococcus. — Its morphology and staining pecu- 



CHARACTERISTICS OF SPECIAL BACTERIA 417 

liarities are given on p. 369. These usually suffice for 
its identification, cultural methods being rarely under- 
taken. In cultures the chief diagnostic point is its failure 
to grow on ordinary media. To grow it the most con- 
venient mediimi is made by adding ascitic or hydrocele 
fluid (which has been obtained under aseptic conditions) 
to melted agar in proportion of i part of serum to 
3 parts of agar. The agar in tubes is melted and 
cooled to about 45° C; the serum is added with a pipet 
and mixed by shaking; and the tubes are again cooled in 
a slanting position. Colonies upon this medium are 
minute, grayish, and translucent. 

7. Diplococcus Intracellularis Meningitidis. — It grows 
poorly or not at all on plain agar. On Loffler's blood- 
serum, upon which it grows fairly well, colonies are round, 
colorless or hazy, flat, shining, and viscid looking. It 
quickly dies out. 

8. Diphtheria Bacillus. — The diagnosis is usually 
made from a study of stained smears from cultures 
upon blood-serum. Its morphology and staining pecu- 
liarities are characteristic when grown on this medium 
(see p. 379). The bacilli are non-motile and Gram- 
positive. The colonies are round, elievated, smooth, 
and grayish. 

9. Typhoid and Colon Bacilli. — These are medium-sized, 
motile, Gram-negative, non-spore-bearing bacilh. Upon 
blood-serum they form rounded, grayish, slightly ele- 
vated, viscid looking colonies, those of the colon bacillus 
being somewhat the larger. They do not liquefy gela- 
tin. They represent the extremes of a large group 
with many intermediate types. They are distinguished 
as follows: 

27 



4l8 BACTERIOLOGIC METHODS 

Typhoid Bacillus. Colon Bacillus. 

Actively motile. Much less active. 

Growth on potato usually invisible. Growth distinctly visible as dirty 

gray or brownish slimy layer. 

No gas produced in glucose media. Produces gas. 

Growth in litmus milk produces no Litmus milk pink and coagulated. 

change. 

Produces no indol in Dunham's Produces indol. (For test, see 

peptone medium. p. 404.) 

Agglutinates with serum from ty- Does not agglutinate with typhoid 

phoid-fever patient. (Recently serum. 

isolated bacilli do not agglutinate 

well.) 

10. Bacillus of Influenza. — Diagnosis will usually rest 
upon the morphology and staining peculiarities, described 
on p. 58, and upon the fact that the bacillus will not grow 
on ordinary media, but does grow upon hemoglobin-con- 
taining media. It can be grown upon agar-slants which 
have been smeared with a drop of blood from a puncture 
in the finger. Before inoculation these slants should be 
incubated to make sure of sterility. The colonies are 
difficult to see without a hand lens. They are very 
minute, discrete, and transparent, resembling small 
drops of dew. 

11. Bacillus of Tuberculosis. — The methods of iden- 
tifying this important organism have been given (pp. 
49 and 168). Cultivation is not resorted to in clinical 
work. It grows very slowly and only on certain media. 
It is Gram-positive and non-motile. 



CHAPTER DC 

PREPARATION AND USE OF VACCINES 

Bacterial vaccines, sometimes called " bacterins," 
which within the past few years have come to play an 
important role in therapeutics, are suspensions of defi- 
nite numbers of dead bacteria in normal salt solution. 
While in many cases, notably in gonorrhea and tuber- 
culosis, ready prepared or " stock " vaccines are satis- 
factory, it is usually desirable and often imperative for 
best results to use vaccines which are especially prepared 
for each patient from bacteria which have been freshly 
isolated from his own lesion. These latter are called 
" autogenous vaccines." Only through them can one 
be certain of getting the exact strain of bacterium which 
is producing the disease. 

I. PREPARATION OF VACCINE 
The method of preparing autogenous vaccines which 
is used in the author's laboratory is here described. 

I. Preparation of Materials. — A number of 2-ounce 
wide-mouthed bottles are cleaned and sterilized. Each is 
charged with 50 c.c. freshly filtered normal salt solution 
(0.85 per cent, sodium chlorid in distilled water), and is 
capped with a new rubber nursing-nipple, without holes, 
inverted as shown in Fig. 162. A small section of hollow 
wire or a hypodermic needle is thrust through the cap 
near the edge to serve as an air vent, and the bottle and 

419 



420 



PREPARATION AND USE OP VACCINES 



contents are sterilized in an autoclave. If an autoclave 
is not at hand, successive steamings in an Arnold steril- 
izer will answer, provided it is not opened between steam- 
ings. After sterilization, the pieces of wire are pulled 
out and the holes scaled with collodion. 






^CTERIALVA^ 







Fig. 162. — \'accine bottles: A, Cap ready to be applied; B, ready for sterilization; 
C, finished vaccine. 



A number of test-tubes, each charged with 10 c.c. of 
normal salt solution and plugged with cotton, are also 
prepared and sterilized. 

2. Obtaining the Bacteria.— A culture on some solid 
medium is made from the patient's lesion, and a pure 
culture is obtained in the usual way. This preliminary 
work should be carried through as quickly as possible. 
If for any reason there is much delay, it is best to begin 
over again, the experience gained in the first trial en- 



PREPARATION OF VACCINE 



421 



abling one to carry the second through more rapidly. 
When a pure culture is obtained, a number of tubes of 
blood-serum or agar — 10 or 1 2 in the case of streptococ- 
cus or pneumococcus, 4 or 5 in the case of most other 
organisms — are planted and incubated over night or 
until a good growth is obtained. 

3. Making the Suspension. — The salt solution from 
one of the 10 c.c. salt-tubes is transferred by means of a 




Fig. 163. — Process of making hermetically sealed capsules. 

sterile pipet to the culture-tubes, and the growth thor- 
oughly rubbed up with a stiff platinum wire or a glass 
rod whose tip is bent at right angles, The suspension 
from the different tubes, usually amounting to about 10 
c.c, is then collected in one large tube (size about 6 by 
I inch) ; and the upper part of the tube is drawn out in 
the flame of a blast lamp or Bunsen burner, as indicated 
in Fig. 163, a short section of glass tubing being fused to 



422 



PREPARATION AND USE OF VACCINES 




the rim of the tube to serve as a 
handle. It is then stood aside, and 
when cool the end is sealed off. 

The resulting hermetically sealed 
capsule is next thoroughly shaken for 
ten to twenty minutes to break up all 
clumps of bacteria. Some small pieces 
of glass or a little clean sterile sand 
may be introduced to assist in this, 
but with many organisms it is not 
necessary. 

4. Sterilization. — The capsule is 
placed in a water-bath at 60° C. for 
forty-five minutes. This can be done 
in an ordinary rice-cooker, with double 
lid through which a thermometer is 
inserted. When both compartments 
are filled with water it is an easy 
matter to maintain a uniform tem- 
perature by occasional application of 
a small flame. The time and tem- 
perature are important : too little heat 
will fail to kill the bacteria, and too 
much will destroy the efficiency of 
the vaccine. 

When sterilization is complete the 
capsule is opened, a few drops are 
planted on agar or blood-serum, and 
the capsule is again sealed. 

5. Counting. — When incubation of 
the plant has shown the suspension to 
be sterile it is ready for counting. 



PREPARATION OF VACCINE 423 

There must be ready a number of clean slides; a few 
drops of normal salt solution on a slide or in a watch- 
glass; a blood-lancet, ' which can be improvised from a 
spicule of glass or a pen; and two slender pipets with 
squarely broken off tips and grease-pencil marks about 
2 cm. from the tip (Fig. 164). These are easily made by 
drawing out a piece of glass tubing, as described on page 

398- _ 

It is necessary to work quickly. After thorough shak- 
ing, the capsule is opened and a few drops forced out 
upon a slide. Any remaining clumps of bacteria are 
broken up with one of the pipets by holding it against 
and at right angles to the slide, and alternately sucking 
the fluid in and forcing it out. The pipet is most easily 
controlled if held in the whole hand with the rubber bulb 
between the thumb and the side of the index-finger. 
A finger is then pricked until a drop of blood appears; 
and into the second pipet are quickly drawn successively : 
I or 2 volumes normal salt solution (or better, a i percent, 
solution of sodium citrate which prevents coagulation); 
a small bubble of air; i volume of blood; a small bubble of 
air; and, finally, i volume of bacterial suspension. (A 
''volume" is measured by the distance from the tip of 
the pipet to the grease-pencil mark.) The contents of 
the pipet are then forced out upon a slide and thoroughly 
mixed by sucking in and out, care being taken to avoid 
bubbles; after which the fluid is distributed to a number 
of slides and spread as in making blood-smears. 

The films are stained with Wright's blood-stain or, 
better, by a few minutes' application of carbol-thionin, 
after fixing for a minute in saturated mercuric chlorid 
solution. With an oil-immersion lens both the red cells 



424 PREPARATION AND USE OF VACCINES 

and the bacteria in a number of microscopic fields are 
counted. The exact number .is not important; for 
convenience 500 red cells may be counted. P'rom the ra- 
tio between the number of bacteria and of red cells, it 
is easy to calculate the number of bacteria in i c.c. of 
the suspension, it being known that there are 5000 million 
red corpuscles in a cubic centimeter of normal human 
blood. If there were twice as many bacteria as red cor- 
puscles in the fields counted, the suspension would con- 
tain 10,000 million bacteria, per cubic centimeter. 

The count can also be made with the hemocytometer, 
using a weak carbol-fuchsin or gentian violet, freshly 
filtered, as diluting fluid. A very thin cover-glass must 
be used; and, after filling, the counting-chamber must 
be set aside for an hour or more to allow the bacteria to 
settle. Mallory and Wright advise the use of the shallow 
chamber manufactured by Zeiss for counting blood- 
plates, but many 2 mm. oil-immersion objectives have 
sufficient working distance to allow the use of the 
regular Thoma counting-chamber, provided a very thin 
cover is used. 

6. Diluting. — The amount of the suspension, which, 
when diluted to 50 c.c, will give the strength desired for 
the finished vaccine having been determined, this amount 
of salt solution is withdrawn with a hypodermic syringe 
from one of the bottles already prepared, and is replaced 
with an equal amount of suspension. One-tenth c.c. of 
trikresol or lysol is finally added and the vaccine is 
ready for use. To prevent possible leakage about the 
cap, the neck of the bottle is dipped in melted paraffin. 
The usual strengths are : staphylococcus. 1000 million in 
I c.c; most other bacteria, 100 million in i c.c. 



DOSAGE 425 

II. METHOD OF USE 

Vaccines are administered subcutaneously, usually in 
the arm or abdominal wall or between the shoulder-blades. 
The rubber cap is sterilized by filling the concavity with 
alcohol for some minutes, usually while the syringe is 
being sterilized. The bottle is then inverted and well 
shaken, when the needle is plunged through the rubber 
and the desired quantity withdrawn. The hole seals 
itself and no collodion is necessary, which is one of the 
advantages of this form of cap. The most satisfactory 
syringe is the comparatively inexpensive " Sub-Q Tuber- 
culin" syringe graduated in hundredths of a cubic centi- 
meter. 

A rapid and efficient technic for giving the injections 
is suggested by Major F. T. Woodbury of the Army 
Medical Corps. The needle is dipped into tincture of 
iodin and some is drawn into the syringe and expelled; 
a small quantity of the vaccine or of sterile water is like- 
wise drawn in and expelled, after which the dose to be 
given is drawn in. The patient's arm is touched with a 
swab of cotton saturated with tincture of iodin and the 
injection is made through the resulting brown spot. 
The syringe is cleaned by drawing into it and expeUing, 
successively, tincture of iodin, alcohol, and air. 

III. DOSAGE 

-Owing to variations, both in virulence of organisms and 
susceptibility of patients, no definite dosage can be 
assigned. Each case is a separate problem. Wright's 
original proposal was to regulate the size and frequency 
of dose by its effect upon the opsonic index, but this is 



426 PREPARATION AND USE OF VACCINES 

beyond the reach of the practitioner. The more widely 
used "clinical method" consists in beginning with a 
very small dose and cautiously increasing until the 
patient shows either improvement or some sign of a 
"reaction," indicated by headache, malaise, fever, ex- 
acerbation of local disease, or inflammatory reaction at 
the site of injection. The reaction indicates that the 
dose has been too large. The beginning dose of staphy- 
lococcus is about 50 million; the maximum, 1000 million 
or more. Of most other organisms the beginning dose 
is 5 million to 10 million; maximum, about 100 million, 
Ordinarily, injections are given once or twice a week; 
very small doses may be given every other day. 

IV. THERAPEUTIC INDICATIONS 

The therapeutic effect of vaccines depends upon their 
power to stimulate the production of opsonins and other 
antibacterial substances which enable the body to com- 
bat the infecting bacteria. Their especial field is the 
treatment of subacute and chronic localized infections, 
in some of which they offer the most effective means of 
treatment at our command. In most chronic infections 
the circulation of blood and lymph through the diseased 
area is very sluggish, so that the antibodies, when formed, 
cannot readily reach the seat of disease. Ordinary 
measures which favor circulation in the diseased part 
should, therefore, accompany the vaccine treatment. 
Among these may be mentioned incisions and drainage 
of abscesses, dry cupping, application of heat. Bier's 
hyperemia, etc., but such measures should not be applied 
during the twenty-four hours succeeding an injection, 
since the first effect of the vaccine may be a temporary 



THERAPEUTIC INDICATIONS 427 

lowering of resistance. Vaccines are of little value, and, 
in general, are contraindicated in very acute infections, 
particularly in those which are accompanied by much 
systemic intoxication, for in such cases the power of the 
tissues to produce antibodies is already taxed to the limit. 
It is true, nevertheless, that remarkably beneficial results 
have occasionally followed their use in such acute condi- 
tions as malignant endocarditis, but here they should 
be tried with extreme caution. 

Probably best results are obtained in staphylococcus 
infections, although pneumococcus, streptococcus, and 
colon bacillus infections usually respond nicely. Among 
clinical conditions which have been treated successfully 
with vaccines are furunculosis, acne vulgaris, infected 
operation- wounds, pyelitis, cystitis, subacute otitis 
media, osteomyelitis, infections of nasal accessory si- 
nuses, etc. Vaccine treatment of the mixed infection is 
doubtless an important aid in tuberculosis therapy, 
and in some cases the result is brilliant. When, as is 
common, several organisms are present in the sputum, 
a vaccine is made from each, excepting the tubercle 
bacillus, 'of which autogeneous vaccines are not used in 
practice. To avoid the delay and consequent loss of 
virulence entailed by study and isolation of the several 
varieties, many workers make the bacterial suspension 
directly from the primary cultures. The resulting vac- 
cines contain all strains which are present in the sputum 
in approximately the same relative numbers. Although 
open to criticism from a scientific standpoint, this 
method offers decided practical advantages in many 
cases. 

It has been shown that vaccines are useful in prevent- 



428 PREPARATION AND USE OF VACCINES 

ing as well as curing infections. Their value has been 
especially demonstrated in typhoid fever. Three or four 
doses of about 50,000,000 typhoid bacilli are given about 
seven days apart. Results in the army, where the plan 
has been tried on a large scale, show that such vaccina- 
tion is effective. 

V. TUBERCULINS 

Tuberculins contain the products of tubercle bacilli 
or their ground-up bodies, the latter class being prac- 
tically vaccines. They are undoubtedly of great value 
in the treatment of localized tuberculosis, particularly 
of bones, joints, and glands; and are of rather indefmite 
though certainly real value in chronic pulmonary tuber- 
culosis, especially when quiescent. The best known are 
Koch's old tuberculin (T. O.), bouillon filtrate (B. F.), 
triturate residue (T. R.), and bacillary emulsion (B. E.). 
There seems to be little difference in the actions of these, 
although theoretically T. R. should immunize against 
the bacillus and B. F. against its toxic products. The 
choice of tuberculin is much less important than the 
method of administration. The making of autogenous 
tuberculins is impracticable, hence stock preparations 
are used in practice. 

Since the dose is exceedingly minute, the tuberculin 
as purchased must be greatly diluted before it is avail- 
able for use. A convenient plan is to use the rubber- 
capped 50 c.c. bottles of sterile salt solution described 
for vaccines (p. 419), adding sufficient tuberculin to give 
the desired strength, together with o.i c.c. trikresol to 
insure sterility. The practitioner should bear in mind 
that while tuberculin is capable of good, it is also capable 



TUBERCULIN IN DIAGNOSIS 429 

of great harm. Everything depends upon the dosage 
and plan of treatment. Probably a safe beginning dose 
for a pulmonary case is 0.0000 1 milligram of B. E., B. F., 
or T. R. ; for gland and bone cases, about o.oooi milligram. 
O. T. is now used chiefly in diagnosis. The intervals are 
about one week or, rarely, three days, when very small 
doses are given. The dose is increased steadily, but with 
extreme caution; and should be diminished or temporarily 
omitted at the first indication of a "reaction," of which, 
in general, there are three forms : 

(a) Getter al: Elevation of temperature (often slight), 
headache, malaise. 

(6) Local: Increase of local symptoms, amount of 
sputum, etc. 

(c) Stick: Inflammatory reaction at site of injection. 

Treatment is usually continued until a maximum dose 

of I mm. is reached, the course extending over a year or 

more. 

VI. TUBERCULIN IN DIAGNOSIS 

The tissues of a tuberculous person are sensitized 
toward tuberculin, and a reaction (see preceding section) 
occurs when any but the most minute quantity of tuber- 
cuUn is introduced into the body. Non-tuberculous 
persons exhibit no such reaction. This is utilized in 
the diagnosis of obscure forms of tuberculosis, the 
test being applied in a number of ways: 

I. Hypodermic Injection. — Koch's old tuberculin is 
used in successive doses, several days apart, of o.ooi , o.oi, 
and 0.1 mg. A negative result with the largest amount 
is considered final. The reaction is manifested by 
fever within eight to twenty hours after the injection. 
The method involves some danger of lighting up a 



430 PREPARATION AND USE OF VACCINES 

latent process, and has been largely displaced by safer 
methods. 

2. Calmette's Ophthalmo-reaction. — One or two drops 
of 0.5 per cent, purified old tuberculin are instilled into 
one eye. Tuberculin ready prepared for this purpose is 
on the market. If tuberculosis exists anywhere in the 
body, a conjunctivitis is induced within twelve to twenty- 
four hours. This generally subsides within a few days. 
The method is not without some, though sUght, risk of 
injury to the eye; and the test is absolutely contraindi- 
cated in the presence of any form of ocular disease, 
A second instillation should not be tried in the same 
eye. 

3. More Reaction. — A 50 per cent, ointment of old tu- 
berculin in lanolin is rubbed into the skin of the abdomen, 
a piece about the size of a pea being required. Dermati- 
tis, which appears in twenty-four to forty-eight hours, 
indicates a positive reaction. The ointment can be 
purchased ready for use. 

4. Von Pirquet's Method. — This is the most satis- 
factory of the tuberculin tests. Two small drops of 
old tuberculin are placed on the skin of the front of the 
forearm, about 2 inches apart, and the skin is slightly 
scarified, first at a point midway between them, and then 
through each of the drops. A convenient scarifier is a 
piece of heavy platinum wire, the end of which is ham- 
mered to a chisel edge. This is held at right angles to 
the skin, and rotated six to twelve times with just suffi- 
cient pressure to remove the epidermis without drawing 
blood. In about ten minutes the excess of tuberculin 
is gently wiped away with cotton. No bandage is neces- 
sary. A positive reaction is shown by the appearance in 



TUBERCULIN IN DIAGNOSIS 43 1 

twenty-four to forty-eight hours of a papule with red 
areola, which contrasts markedly with the small red spot 
left by the control scarification. 

These tests have very great diagnostic value in chil- 
dren, especially those under three years of age, but are 
often misleading in adults, positive reactions occurring 
in many apparently healthy individuals. Negative tests 
are very helpful in deciding against the existence of 
tuberculosis. 



APPENDIX 

L APPARATUS, REAGENTS, AND STAINS 

The apparatus and reagents listed here are sufficient 
for all but the rarer tests described in the text. Those in 
smaller type are less frequently required. For ordinary 
routine work a much smaller list will suffice. 

A, APPARATUS 

Beakers and flasks, several sizes, preferably of Jena 
glass. 

Blood lancet, or some substitute (Fig. 68). 

Bunsen-burner or alcohol lamp. 

Buret, 25 c.c. capacity, preferably with Schellbach 
stripe. 

Buret and filter-stand combined. 

Centrifuge — hand, electric, or water-power (Figs. 20 
and 21). With the last two a speed indicator is desirable. 
Radius of arm when in motion should be 6f inches. 
Plain and graduated tubes accompany the instrument ; 
milk- tubes (Fig. 157) must be purchased separately. The 
hematocrit attachment (Fig. 77) is not much used. 

Cigaret-paper, "Zig-zag" brand or some similar thin 
paper, for making blood-films. 

Corks, preferably of rubber, with one and two holes. 

Cover-glasses, No. 2 thickness — |-inch squares are 
most convenient. 

Cover-glass forceps. 

432 



APPARATUS 433 

Esbach's tube (Fig. 27). 

Evaporating dish. 

Filter-paper: ordinary cheap paper for urine filtration; 
"ashless" quantitative filter-paper for chemic analyses. 

Glass funnels. 

Glass rods and tubing of sodium glass : for stirring rods, 
urinary pipets, etc. 

Glass slides: the standard i- by 3-inch size will answer 
for all work, although a few larger slides will be found 
convenient; those of medium thickness are preferable. 

Graduates, cylindric form, several sizes. 

Graniteware basin. 

Hemoglobinometer : see pp. 185 to 191 for descriptions 
of the different instruments. 

Hemocy tometer : either Tiirck or Zappert ruling is 
desirable (Figs. 73, 74, and 79). 

Hypodermic syringe: the "Aseptic Sub-Q, Tubercu- 
lin," is probably the most useful type. 

Incubator (p. 397). 

Labels for slides and bottles. 

Litmus-paper, red and blue, Squibb's preferred. 

Mett's tubes (pp. 300 and 399). 

Microscope (Fig. i). Equipment described on p. 31. 

Petri dishes. 

Platinum wires (p. 397). 

Sterilizers: the Arnold type for steaming; oven for dry 
sterilization (p. 396). 

Stomach-tube. 

Test-glass, conic jOne side painted half white, half black . 

Test-tubes, rack, and cleaning brush. 

Ureometer, Doremus-Hinds' pattern (Fig. 24). 

Urinometer, preferably Squibb's (Fig. 17). 

28 



434 APPENDIX 

Blood-fixing oven, or Kowarsky's plate (Fig. 84). 

Copper-foil and gauze. 

Cotton, absorbent, for filtering, etc. 

" Cotton-batting " for plugging tubes. 

Culture-media. The selection depends upon the work 
to be done (p. 401). 

Holt's cream gage and hydrometer (Fig. 156). 

Horismascope (Fig. 26). 

Pipets, graduated, 5 to 50 c.c. capacity. 

Ruhemann's tube for uric-acid estimation (Fig. 25). 

Saccharimeter (Fig. 29). 

Strauss' separatory funnel for lactic-acid test (Fig. 105). 

Suction filter. 

Urinopyknometer of Sa.xe (Fig. 18). 

Widal reaction outfit: either living agar cultures of the 
t>-phoid bacillus, or the dead cultures with diluting apparatus, 
which are sold under various trade names. 

Water-bath. 

B. REAGENTS AND STAINS 

All stains and many reagents are best kept in small 
dropping bottles. Formulae are given in the text. Dry 
stains (Griibler's should be specified) and most staining 
solutions and chemical reagents can be purchased of 
Bausch & Lomb Optical Co., Rochester, New York, or 
Eimer & Amend, New York. For the physician who 
does only a small amount of work, the " Soloid " tablets 
manufactured by Burroughs. Wellcome & Co. are con- 
venient and satisfactory. These tablets have only to 
be dissolved in a specified amount of fluid to produce 
the finished stain. IMost of the stains mentioned here 
come in this form. 

Acid, glacial acetic. Other strengths can be made 
from this as desired. 



REAGENTS AND STAINS 435 

Acid, hydrochloric, concentrated (contains about 32 
per cent, by weight of absolute hydrochloric acid). 
Other strengths can be made as desired. 

Acid, nitric, strong, colorless. 

Acid, nitric, yellow. Can be made from colorless acid 
by adding a splinter of pine, or allowing to stand in 
sunlight. 

Acid, sulphuric, concentrated. 

Alcohol, ethyl (grain-alcohol) . This is ordinarily about 
93 to 95 per cent., and other strengths can be made as 
desired. 

Aqua ammoniae fortior (sp. gr. 0.9). 

Bromin, or Rice's solutions (p. 95), for urea estimation. 

Chloroform. 

Diluting fluid for erythrocyte count (p. 198). 

Diluting fluid for leukocyte count (p. 213). 

Dimethyl-amido-azobenzol, 0.5 per cent, alcoholic 
solution. 

Distilled water. 

Esbach's or Tsuchiya's reagent (p. 105). 

Ether, sulphuric. 

Ferric chlorid: saturated aqueous solution and 10 per 
cent, aqueous solution. 

Haines' (or FehHng's or Benedict's) solution (pp. 109, 
no). 

Lugol's solution {Liquor lodi Compositus, U. S. P.). 
Gram's iodin solution (p. 57) can be made from this by 
adding fourteen times its volume of water. 

Obermayer's reagent (p. 91). 

Phenylhydrazin, pure. 

Phenol. 

Phenolphthalein, i or 0.5 per cent, alcoholic solution. 



436 APPENDIX 

Purdy's (or Fehling's or Benedict's) solution (pp. 112- 

115). 

Robert's reagent (p. 103). 

Sodium chlorid (table salt), saturated aqueous solu- 
tion. 

Sodium hydroxid (caustic soda) , 40 per cent, solution ; 
other strengths can be made from this as desired. 

Sodium hydroxid, decinormal solution. The prac- 
titioner will find it best to purchase this solution ready 
prepared. Eimer and Amend, New York, and many 
other chemical supply houses carry it in stock. For 
ordinary cUnical work 41 grams of Merck's " sodium 
hydrate by alcohol " from a freshly opened bottle may 
be dissolved in icxx) c.c. water. This makes a normal 
solution and must be diluted with 9 volumes of water to 
make the decinormal solution. 

Sodium nitrite, 0.5 per cent, solution for diazo-reaction. 
Must be freshly prepared. 

Sulphanilic acid solution for diazo-reaction (p. 128). 

Stains: 

Carbol fuchsin (p. 50). 

Eosin, saturated aqueous solution. 

Formalin - gentian - violet, or anihn - gentian - violet 

(P- 57)- 
Gabbet's stain or Pappenheim's methylene-blue 

stain (p. 51). 
Loffler's alkaline methylene-blue solution (p. 57). 
Pappenheim's pyronin-methyl-green stain (p. 408). 
Stain for fat: Sudan III. saturated solution in 

70 per cent, alcohol; or i per cent, aqueous 

solution osmic acid. 
Wright's or Harlow's stain for blood. 



REAGENTS AND STAINS 437 

Tincture of guaiac, diluted to a light sherry-wine color 
(keep in a dark glass bottle). 
Turpentine, "ozonized" (p. 125). 

Acid, boric, for preserving urine (p. 69). 

Acid, oxalic. 

Acid, salicylous (salicyl aldehyd), 10 per cent, alcoholic 
solution. 

Alcohol, amylic. 

Alcohol, ethyl, absolute. 

Alcohol, methyl (pure). 

Antiformin (p. 52). 

Barium chlorid mixture (p. 89). 

Benzol. 

Boas' reagent or Gunzburg's (p. 290). 

Boggs' reagent (p. 387). 

Calcium chlorid, i per cent, solution. 

Canada-balsam in xylol: necessary only when permanent 
microscopic preparations are made. 

Carbon disulphid. 

Charcoal, animal. 

Chromium trioxid. 

Congo-red, strong alcoholic solution. 

Copper sulphate. 

Diluting fluid for blood-platelet count (pp. 215, 216). 

Egg-albumen discs in glycerin (p. 293). 

Ether, acetic, pure, 

Florence's reagent (p. 393). 

Formalin (40 per cent, solution of formaldehyd gas). 
• India-ink (Gunther and Wagner) (p. 391). 

lodin crystals. 

Iron sulphid. 

Lead acetate (sugar of lead) ; used in 10 per cent, solution 
to clarify urine. 



438 APPENDIX 

Lead acetate, tribasic. 
Lime-water. 

Miiller's fluid saturated with mercuric chlorid (p. 56). 
Orcin. 

Pepsin, U. S. P. 
Phenylhydrazin hydrochlorid. 
Potassium ferrocyanid, 10 per cent, solution. 
Potassium oxalate (neutral). 
Potassium persulphate. 
Ruhemann's reagent (p. 97). 

Silver nitrate crystals; also dram to the ounce aqueous 
solution, and " ammoniated " solution (p. 97). 

Sodium alizarin sulphonate, i per cent, aqueous solution. 
Sodium carbonate. 

Sodium chlorid, 2 per cent, solution ; from this normal salt 
solution (0.8 per cent.) can be made as desired. 
Sodium hyposulphite. 
Sodium nitroprussid. 
Sodium sulphate. 
Stains: 

Bismarck-brown, saturated aqueous or alcoholic solution. 

Carbol-thionin. 

Ehrlich's triple stain for blood. 

Eosin, 0.5 per cent, alcoholic solution for blood. 

Fuchsin, weak solution; can be made when desired by 
adding a little carbol-fuchsin to a test-tube of water. 

Gentian- violet, saturated alcoholic solution. 

Giemsa's stain (p. 390). 

Methylene-blue and borax solution (p. 254). 

Methylene-blue, saturated aqueous solution for blood. 

Van Gieson's stain for Negri bodies (p. 395). 
Sulphur, powdered. 

Talc, purified (Talcum Purificatum, U. S. P.). 
Trichloracetic acid solution (p. 102). 



WEIGHTS AND MEASURES 



439 



Uranium nitrate, 5 per cent, aqueous solution. 

Xylol. 

Zinc, arsenic free. 



IL WEIGHTS, MEASURES, ETC, WITH 
EQUIVALENTS 



Meter (unit of length) : 

Gram (unit of weiglit) : 
Liter (unit of capacity) : 



METRIC 

Millimeter (mm.) = j^bb meter. 
Centimeter (cm.) = yjg meter. 
Kilometer = looo meters. 



Micron (/x) 



= xiAiff millimeter. 



Milligram (mg.) = ^o^jj gram. 
Kilogram (kilo.) = looo meters. 

Cubic Centimeter = jo'jj liter. Same measure as milli- 
liter (ml.). 



I Millimeter = 
I Centimeter ^ 
I Meter = 



0-03937 (5's approx.) in. 
1000 microns. 
0.3937 (S approx.) in. 
0.0328 feet. 

39-37 in- 
3.28 feet. 
!b4d 



X Micron (M)={^^7^"aU^,t„, 



I Sq. Millimeter = 0.00155"! 

1 Sq. Centimeter = 0.1550 >sq. in. 

1 Sq. Meter = 1550 j 

1 Sq. Meter = 10.76 sq. feet. 



1 Inch = 25.399 millimeters. 

I Sq. Inch = 6.451 sq. centimeters. 
1 Cu. Inch = 16.387 cu. centimeters. 



I Gram 



I Kilogram = 



X Liter 



15-43 grams. 

0.563 dram "j 

0.035 ounce > Avoir. 

0.0022 pound) 

0.257 dram l 

0.032 ounce >Apoth. 

0.0027 pound 1 

35.27 ounce (Avoir.). 

2.2 pound (Avoir.), 
f 1.056 (i approx.) quart. 
^■< 61.02 cu. inches. 
(1000 cu. centimeters. 



I Cu. Millimeter = 0.00006 ) . 
1 Cu. Centimeter = 0.0610 J " 

1 Cu. Centimeter = o.ooi liter. 

,.32 cu. feet. 

1025.4 cu. iu. 



I Cu. Meter 



135-3 
\6io: 



I Foot = 30.48 centimeters. 
I Sq, Foot = 0.093 sq. meter. 
I Cu. Foot = 0.028 cu. meter. 



AVOIRDUPOIS WEIGHT 



' Ounce = {437.5^^f- 
1 Pound = 16 oimces. 



I Grain = 0.065 (^ approx.) "I 

I Dram = 1.77 (ij approx.) 1 _._s 

I Ounce = 28.35 (30 approx.) f° 

I Pound = 453-59 (500 approx.) J 

I Pound = 27.7 cu. inches. 

1 Pound = 1.2151b. Troy. 



APOTHECARIES' MEASURE 



I Dram = 60 minims. 
1 Ounce = 8 drams. 
'I Pint = 16 ounces. 
I Gallon = 8 pints. 



I Dram = 3.70 

I Ounce = 29.57 

1 Pint = 473.1 

X Gallon = 3785.4 

I Gallon = 231 cu. inches 



cu. centimeters. 



440 



APPENDIX . 



APOTHECARIES' WEIGHT 



I Scrup 


e = 


= 20 gra 


ns. 




I Grair 


= 


- 0.065 1 


I Dram = 3 scruples = 60 


grains 


I Uram = 3 887 1 „„„, 
I Ounce =3110 [^a""*- 
I Pound =^ 373.2 J 


I. Ounce = 8 drams -= 480 grains. 


I Pound -— 12 ounces. 




To convert minims 


into cubic centimeters multiply by 0.061 
" cubic centimeters " 29.57 


" jluidounces 


" " grains 


" 


grams " " 0.0648 


" " drams 


" 


grams " " 3.887 


" " cubic centimeters 




minims " " 16.23 


" " cubic centimeters 




jluidounces " " 0.0338 


" " grams 




grains " " 15.432 


" " grams 




drams " " 0.257 


TEMPERATURE 


Centigrade. Fahrenheit. 


Centigrade. Fahrenheit. 


110° 230° 




37° 98.6° 


100 










212 




36.5 






• • 97-7 


95 










203 




36 






. , 96.8 


90 










194 




35-5 • 






• • 959 


85 










185 




35 






• • 95 


80 










176 




34 






• 93-2 


75 










167 




33 






• • 914 


70 










•158 




32 






89.6 


65 










149 




31 






. . 87.8 


60 










140 




3" 






. . 86 


55 










131 




25 






• • 77 


50 










122 




20 






. . 68 


45 










"3 




15 






• ■ 59 


44 










I I 1.2 




10 






• ■ 50 


43 










109.4 




+5 






. . 41 


42 










107.6 











• 32 


41 










105.8 




—5 






. . . 23 


40.5 










104.9 




— 10 






. . 14 


40 










104 




— 15 




• ■ 45 


39-5 










103. 1 
102.2 




— 20 — 4 


39 




38.5 










IOI.3 




0.54° =- 1° 


■ 38 










100.4 




I = 1.8 


37-5 










99-5 




2 = 3.6 
















25 






= 4-5 



To convert Fahrenheit into Centigrade, subtract 32 and 
multiply by 0.555. 

To convert Centigrade into Fahrenheit, multiply by 
1.8 and add 32. 



INDEX 



Absorption, toxic, degree of, 236 
Absorptive power of stomach, 306 
Accidental albuminuria, 100 
Acetanilid in urine, 132 
Acetic acid in gastric contents, 291 
Acetone in urine, 118. See also 

Acetomiria. 
Acetonuria, 118 

after anesthesia, 119 

detection, 119 

Frommer's test in, 122 

Gunning's test in, 120 

Lange's test in, 121 

Legal's test for, Lange's modifi- 
cation, 121 

Lieben's test in. Gunning's modi- 
fication, 121 

tests, 1 19-122 
Achard and Castaigne's methylene- 

blue test for urine, 79 
Achlorhydria, 297 
Acholic stools, 312 
Achromatic objectives, 23 
Achylia gastrica, gastric contents 

in, 304 
Acid deficit of gastric contents, 299 

intoxication, cause, 118 
Acid-fast bacilli, 53 
Acidity of urine, quantitative esti- 
mation, 74 
Folin's method, 74 
Acidophilic structures of blood, 221 
Actinomyces bovis in sputum, 46 
Agar-agar, glycerin, preparation of, 
402 

preparation of, 402 
Agglutination, 257 
Agglutinins, 257 
Air-bubbles in urine, 173 
Albumin in sputum, 63 



Albumin in urine, 99. See also 

Albuminuria. 
AJbuminometer, Esbach's, 105 
Albuminuria, 99, 100 

accidental, 100 

centrifugal estimation of albu- 
min, 106 

cyclic, 100 

detection, 102 

Esbach's estimation of albumin, 

estimation of albumin in, quan- 
titative, 105 

false, 100 

from blood changes, loi 

from kidney changes, loi 

heat and nitric acid test in, 104 
test in, Purdy's, 104 

nitric acid test in, 104 

orthostatic, 100 

physiologic, 100 

postural, 100 

Purdy's centrifugal method, 106 
heat test in, 104 
table after. centrifugation, 107 

renal, 100 

Robert's test in, 103 

tests, 102-106 

trichloracetic acid test in, 102 

Tsuchiya's estimation of albu- 
min, 105 
Alkaline methylene-blue, LoflSer's, 

57 
phosphates in urine, 87 
urine, unorganized sediments in, 
148 
Alkapton bodies in urine, 1 26 
Alkaptonuria, 126 
Alveolar cells in sputum, 62 
Amboceptor, 265, 266, 267 

441 



442 



INDEX 



Amboceptor, hemolytic, 269 
Ameba?, 328. See also Entamceba. 
Ameboid movements of malarial 

parasites, 248 
Amidobenzol test for free hydro- 
chloric acid, 290 
Ammonia in urine, 97 
Brown's test. 99 
decreased, 98 

estimation, quantitative, 98 
increased, 98 

Ronchese-Malfatti formalin 
test, 98 
Amnioniated silver nitrate solution, 

97 
Ammoniomagnesium phosphate 

crystals in urine, 148 
Ammonium urate crystals in urine, 

151 
Ammon's horn, 394 
Amoeba histolytica in sputum, 48 
Amorphous phosphates in urine, 72, 

87, 149 
in mass, 160 

urates in urine, 72, 143 
in mass, 160 
Anaemia infantum pseudoleukaem- 

ica, 282 
Anemias, 275 

aplastic, 279 

blood-picture in, 276 

blood-plaques in, 214 

color index in, 200 

degeneration of Grawitz in, 228 

erythroblasts in blood in, 230 

erythrocytes in, 226 

leukopenia in, 202 

lymphocytes in, 234 

myelocj'tes in, 242 

oligocythemia in, 193 

pernicious, 277 

p)olychromatophilia in, 227 

primary', 277 

secondary-, 276 

splenic, 280 
Anesthesia, acetonuria after, 119 
Angina, \'incent's, 379 
spirochaete of, 331 
Anguillula, 354 

aceti, 354 
in urine, 171 
Anilin dyes for blood-films, 220 



Anilin-gentian- violet stain, 57 

Animal inoculation, 375 

method for tubercle bacillus in 

sputum, 53 
of bacteria, 415 
parasites, 323. See also Para- 
sites, animal. 

Anopheles, 248, 250 

Antibodies, 265, 269 

Antiformin method for bacillus 
tuberculosis in sputum, 52 

Antigen, 269 

Antipyrin in urine, 132 

Anuria, 71 

Aplastic anemia, 279 

Apochromatic objectives, 23 

Apothecaries' measure, 439 
weight, 440 

Apparatus, 396, 432 

Appendicitis, leukocytosis in, 206 

Arsenic in urine, 133 

Gutzeit's test for, 133 
Reinsch's test for, 133 
poisoning, anemia from, 276 

Arthropoda, 366 

Ascaris. 354 
lumbricoides, 354 
ova of, 355 

Asparagus, odor of urine from, 73 

Asthma, bronchial, eosinophilia in, 

239 
sputum in, 66 
Atrophic gastritis, gastric contents 

in, 305 
Atropin in urine, 133 
Autoclave, 396 
Autogenous vaccines, 419 
Avoirdupois weight, 439 



Babcock estimation for fat in milk, 

386^ 
Babesia, 339 
bigeminum, 339 
hominis, 339 
Bacillus, acid-fast, 53 

Boas-Oppler, in feces, 318 

in gastric contents, 303 
colon, 417 

in otitis, 383 
diphtheria, 417 

in eye affections, 382 



INDEX 



443 



Bacillus, diphtheria, in mouth, 378 
Koch-Weeks, in conjunctivitis, 

381 
mucosus capsulatus in sputum, 

of Friedlander in otitis, 383 

in sputum, 58 
of influenza, 418 

in spinal fluid, 376 

in sputum, 58 
of Vincent's angina, 380 
pyocyaneus in otitis, 383 
smegma, 53, 169 
tuberculosis, 418 

in cerebrospinal fluid, 374 

in feces, 319 

in otitis, 383 

in pus, 369 

in sputum, 36, 49 
animal inoculation method, 

53 
antiformin method, 52 
examination, 36 
Gabbet's method, 49 
methods for, 49-53 
Pappenheim's method, 51 
Ziehl-Neelsen method, 51 
in urine, 168 
typhosus, 417 
in blood, 244 
technic, 245 
in urine, 168 
xerosis in eye, 382 
Bacteria, animal inoculation, 415 
characteristics of, 415 
collection of material for cultural 

examination, 412 
cultural methods of examining, 

412 
cultures of, study, 413 
Gram-negative, 409 
Gram-positive, 409 
in blood, 244 
in feces, 318 

stains for, 318, 319 
- in gastric contents, 303 
in milk, 384 
in pus, 367 
in sputum, 49 
in urine, 72, 167 
incubation of, 413 
inoculating media for, 412 



Bacteria, methods of studying, 412 
microscopic examination, 412 
obtaining of, in preparation of 

vaccines, 420 
stains for, 407 

for morphology, 407 
Bacterial casts in urine, 159 

vaccines, 419. See also Vaccines. 
Bacterins, 419. See also Vaccines. 
Bacteriologic methods, 396 

study of blood, 244 
Bacteriolysis, 265 
Balantidium, 339 

coli, 339 
Basket-cells, 243 

Basophilic granular degeneration, 
227 
leukocytes, 240 
structures of blood, 220 
Bass and Watkins' method for 

Widal reaction, 261 
B. E. tuberculin, 428 
Beef extract bouillon, preparation 
of, 401 
infusion, preparation of, 401 
tapeworm, 347 
Bence-Jones' body, 108 

detection, 108 
Benedict's estimation of glucose in 
urine, 115 
test for glucose, no 
B. F. tuberculin, 428 
Bial's orcin test for pentoses, 118 
Bile acids in urine, 124 
Hay's test, 124 
tests, 124 
diminished flow, indican in urine 

from, 91 
in feces, 315 
in gastric contents, 289 
in urine, 123 
Gmelin's test for, 123 
Smith's test for, 123 
tests, 123 
medium, preparation of, 405 
Bile-pigment in urine, 123 
Bilharzia haematobia, 344 
Bilharziasis, 344 
Bilifuscin in urine, 123 
Biliousness, indican in urine in, 90 
Bilirubin in feces, 315 
in urine, 123 



444 



INDEX 



Biliverdin in urine, 123 
Black sputum, 39 
Bladder, hemorrhage from, 166 
schistosomum haematobium as 
cause, 167 
Blepharoconjunctivitis, 381 
Blepharoplast of tr>panosome, 53:^ 
Blood, 180 

acidophilic structures of, 221 
amount of, total, 181 
animal parasites in, 247 
bacillus typhosus in, 244 

technic, 245 
bacteria in, 244 
bactcriologic study of, 244 
basophilic structures of, 220 
changes, albuminuria from, loi 
in blood diseases, table, 283 
coagulation of, 181 
prevention, 182 
time, 181, 182 

Bogg's method of estimating, 
182 
color index, 200 
color of, 181 
constituents, 180 
count, Gibson's chart for, 236, 

.237 
diseases, blood changes in, table, 

283 
Ehrlich's triple stain for, 221 
embryos of trichinella spiralis in, 

257 
eosin and methylene-blue for, 221 
eosinophilic structures of, 221 
erythrocj'tes in, number, 192 
filarial embryos in, 256 
guaiac test for. 274 
Harlow's stain for, 224 
hemin test for, 274 
in anaemia infantum pseudoleu- 

kaemica, 282 
in anemia, 276 

aplastic, 279 

pernicious, 277 

secondar>-, 276 
in aplastic anemia, 279 
in chlorosis. 279 
in feces, 312, 314, 318 
in gastric contents, 289, 295, 302 

test for, 295 
in Hodgkin's disease, 282 



Blood in leukemia, 280 
in l>Tnphatic leukemia, 281 
in myelogenous leukemia, 280 
in pernicious anemia, 277 
in pseudoleukemia, 282 
in splenic anemia, 280 
in urine, 72, 166 
increase of, 184 
leukocytes in, number, 202 
malarial parasites in, 248. See 

also Malarial parasites. 
neutrophilic structures of, 221 
obtaining of, for coagulation test, 

183 
oxiphilic structures of, 221 
parasites, 244 
pathology, special, 275 
polychrome methylene-blue eosin 

stains for, 222 
reaction of, 181 
recognition of, test for, 274 
spirochseta recurrentis in, 247 
stained, study of, 216 
stains, 216 

for films, 216, 219 
Teichmann's test for, 274 
triple stain for, Ehrlich's, 221 
trj'panosoma gambiense in, 247 
typhoid bacilli in, 244 

technic. 245 
unstained, malarial parasites in, 

253 
volume index, 200 

Larrabee's method, 201 
method, 201 
watery, 181 
Wright's stain for, 222 
Blood-casts in urine, 158 
Blood-corpuscles in feces, 318 
red, 180 
decrease of, 192. See also 

Oligocythemia. 
in gastric contents, 302 
in sputum, 63 
in urine, 165 

increase of, 192. See also 
Polycythemia. 
white, 180 
Blood-dust of Miiller, i8i 
Blood-films, 216 
anilin dyes for, 220 
chemic fixation of, 219 



INDEX 



445 



Blood-films, cigarette-paper meth- 
od for, 218 

drying, 219 

Ehrlich's two cover-glass method, 
217 

fixing, 219 

heat fixation for, 219 

Kowarsky's plate for fixing, 219 

making, 216 

malarial parasites in, 254 

spreading, 216 

stained, study of, 225 

staining, 216, 220 

stains for, 216, 220 

two-slide method, 217 
Blood-lancet, Daland's, 183 
Blood-plaques, 180 

enumeration, 213 

in anemia, 214 

in infections, 214 

in leukemia, 214 

in purpura haemorrhagica, 214 

Kemp-Calhoun-Harris estima- 
tion, 214 

stained, study of, 243 

variations in numbers, 213 

Wright and Kinnicutt's estima- 
tion, 216 
Blood-platelets, 180. See also 

Blood- plaques. 
Blood-serum, 181 

LofHer's preparation of, 403 

reactions, 257 
Boas' reagent, 291 

test for free hydrochloric acid, 290 

test-breakfast, 286 
Boas-Oppler bacillus in feces, 318 

in gastric contents, 303 
Bodies, Cabot's ring, 230 

Leishman-Donovan, 335 
Bodo, 336 

urinarius, 336 
Body, Bence-Jones', 108 

detection, 108 
Boggs' coagulation instrument, 182 

method of estimating coagulation 
time of blood, 182 

modification of Esbach method 
for proteins in milk, 387 

reagent, 387 

throttle control for blood-count- 
ing pipet, 209, 210 



Boil, Delhi, Leishmania tropica of, 

335 

Bordet and Gengou test in ty- 
phoid fever, 267 

Boston's method for keeping semen 
for examination, 391 

Bottles, vaccine, 420 

Bouillon, beef extract, preparation 
of, 401 
infusion, preparation of, 401 

Brick-dust deposit in urine, 72, 143 

Bromids in urine, 133 

Bronchi, cylindric cells from, in 
sputum, 61 

Bronchial asthma, eosinophilia in, 

239 
sputum in, 66 
Bronchiectasis, sputum in, 65 
Bronchitis, sputum in, 64 
Brown's test for ammonia in urine, 

99 
Bubbles of air in urine, 173 
Buerger's method for pneumo- 
coccus capsules, 55 
in pus, 368 
Butyric acid in gastric contents, 291 



Cabot's classification of pathologic 
polymorphonuclear leukocy- 
tosis, 206 
ring bodies, 230 
Calcium carbonate in iirine, 150 
oxalate in urine, 144 
phosphate crystals in urine, 149 
Calculus in feces, 313 
renal, urine in, 177 
vesical, urine in, 178 
Calmette's ophthalmo-tubercuHn 

reaction, 430 
Cammidge's pancreatic reaction, 
129 
technic, 129 
Capsules of pneumococcus, Buer- 
ger's method for, 54 
Carbol fuchsin, 50 

thionin stain for bacteria, 408 
Carcinoma, gastric, bacteria in 
feces in, 318 
gastric contents in, 305 
Casts, fibrinous, in sputum, 45 
in urine, 152 



446 



INDEX 



Casts in urine, negative-staining, 
154 

Catarrh, vernal, eosinophilic leuko- 
cytes in, 383 , , 

Cedar oil for oil-immersion objec- 
tive, 24 

Cells, alveolar, in sputum, 62 
basket-, 243 

body-, ciliated, in sputum, 48 
cylindric, in sputum, 61 
eosinophilic, in sputum, 60 
epithelial, in sputum, 61 

in urine, 162, 163 
heart-failure, in sputum, 41, 62, 

. 65 

in sputum, 59 
stains for, 59 

mast-, 240 

polyhedral, in urine, 162 

shadow, in urine, 166 

squamous, in sputum, 61 

vegetable, in feces, 316 

yeast-, in urine, 171 
Centigrade and Fahrenheit scales, 

440 
Central illumination of microscope, 

19 . 
Centrifuge for albumin in urine, 106 
for chlorids in urine, 83 

Purdy's table, 86 
for sulphates in urine, Purdy's, 89 
Purdy's, 83 
tubes, Purdy's, 85 
water-motor, 84 
Cercomonas, 335 

hominis, 336 
Cerebrospinal fever, epidemic, cere- 
brospinal fluid in, 374 
fluid, bacillus tuberculosis in, 374 
examination, 374 
Cestoda, 341, 345 
Cestodes, 341. 345 
Charcot-Leyden crystals in feces, 
320 
in sputum, 44, 45 
Chart, Gibson's, 236, 237 
Chemic examination of sputum, 63 

fixation of blood-films, 219 
Chemotaxis, 203 
Chlorids in urine, 82 

estimation, Purdy's centrifugal 
methods, 83 



Chlorids in urine, estimation, 
Purdy's table, 86 
quantitative, 83, 85 
in nephritis, 82 
Chlorosis, 279 
color index in, 200 
leukopenia in, 202 
lymphocytes in, 234 
oligocythemia in, 193 
Cholesterin crystals in sputum, 45 
Chrysomyia macellaria, 366 
Chyluria from filaria infection, 148 
Cigarette-paper method for blood- 
films, 218 
Cilia, 328, 339 

Ciliated body-cells in sputum, 48 
Cirrhosis of liver, anemia from, 276 
Coagulation, 181 

instrument, Boggs', 182 
prev'ention of, 182 
time, 181, 182 
Boggs' method of estimating, 
182 
Coccidium, 338 
cuniculi, 338 
Cochin China diarrhea, 362 
Coffin-lid crystals in urine, 148 
Colon bacillus, 417 

in otitis, 383 
Color index of blood, 200 
in chlorosis, 200 
in pernicious anemia, 200 
of blood, 181 
Combined hydrochloric acid, 284 
Complement, 266, 267, 269 

deviation, 268 
Concretions in feces, 313 
Condenser for microscope, 22 
Congo-red test for free acids in gas- 
tric contents, 290 
Conjugate sulphates, 90 
Conjunctivitis, acute infectious, 381 
bacteria of, 381 
blepharo-, 381 
diphtheritic, 382 
gonorrheal, 382 
pseudomembranous, 382 
Cook's method for purin bodies, 96 
Corpuscles, blood-, in feces, 318 
red, 180 
decrease of, 192. See also 
Oligocythemia. 



INDEX 



447 



Corpuscles, blood-, red, in gastric 
contents, 302 
in sputum, 63 
in urine, 165 

increase of, 192. See also 
Polycythemia. 
white, 180 
pus-, 236, 367 
in feces, 318 
in gastric contents, 302 
in sputum, 59 
in urine, 163 
suspension in Wassermann reac- 
tion, 269 
Corrections for objectives, 24 
Cotton fibers in urine, 161, 172 
fibrils in sputum, 42 
sterilization of, 400 
Cows' milk, 384 
Croupous pneumonia, sputum in, 

66 
Cryoscopy of urine, 79 
Crystals, Charcot-Leyden, in spu- 
tum, 44, 45 
in feces, 319 
in sputum, 45 
Culex, 250 
Cultural methods of examining 

bacteria, 412 
Culture-media, 401 
preparation of, 401 
reaction of, 405 
sterilization of, 399 
tubing, 406 
Cultures of bacteria, study, 413 
Culture-tubes, 397 

preparation of, 400 
Curds in feces, 314 

of milk in feces, 317 
Curschmann's spirals in sputum, 43 
Cyclic albuminuria, 100 
Cylindric cells in sputum, 61 
Cylindroids in urine, 160 
Cysticercus cellulosse, 348 
Cystin crystals in urine, 146 
Cystinuria, 146 
Cystitis, urine in, 178 
Cysts, daughter-, 349 
Cytodiagnosis, 372 

Daland's blood-lancet, 183 
hematocrit, 201 



Dare's estimation of hemoglobin, 
189 
hemoglobinometer, 189 

Dark ground illumination of mi- 
croscope, 21 

Daughter-cysts, 349 

Definitive host of animal parasites, 

324 
Degeneration of Grawitz, 227 
Delhi boil, Leishmania tropica of, 

335 
Demodex folliculorum, 366 
Desmoid test, Sahli's, of gastric di- 
gestion, 308 
Dextrose in urine, 108. See also 

Glycosuria. 
Diabetes insipidus, urine in, 179 

mellitus, urine in, 1 79 
Diacetic acid in urine, 122 

Gerhardt's test for, 122 
Lindemann's test for, 122 
tests, 122 
Diarrhea, Cochin China, 362 

polycythemia in, 192 
Diazo reaction, 126 
in measles, 128 
in tuberculosis, 127 
in typhoid fever, 127 
technic, 128 
substances in urine, 126 
Dibothriocephalus, 351 
latus, 345, 351 
anemia from, 276 
infection with, decrease of he- 
moglobin from, 185 
ova of, 352 
Dicroccelium, 342 
lanceatum, 342 
Diet, Schmidt's, for examination of 

feces, 320 
Digestion, gastric, Sahli's test for, 

308 
Digestive leukocytosis, 205 
Dilatation of stomach, gastric con- 
tents in, 304 
Diluting fluids for blood count, 198 
in leukemia, 213 
for blood-plaque count, 215 
Dilution in preparation of vaccines, 

424 
Diphtheria bacillus, 417 
of nasopharynx, 378 



448 



INDEX 



Diphtheria of tonsils, 378 
Diphtheritic conjunctivitis, 382 
Di()lobacillus of Morax and Axen- 

fcld, 381 
Diplococcus intracellularis menin- 
gitidis, 374, 417 

of Friinkcl in sputum, 54 
DipyHdium, 351 

caninum, 351 
Distilling apparatus, 120 
Dittrich's plugs in sputum, 3Q 
Donne's test for pus in urine, 72 
Doremus-IIinds' ureometer, 94 
Dosage of tuberculin, 428 

of vaccines, 425 

clinical method, 426 
Dourine, trypanosoma equiperdum 

oi, 335 
Drugs, effect of, on urine, 71, 132 

leukocytosis from, 207 

resinous, in urine, 137 
Drunkard's pneumonia, sputum in, 

39 
Dry objective, 24 
Dumb-bell crystals in urine, 150, 

151 

Dunham's peptone solution, prep- 
aration of, 404 

Dwarf tapeworm, 350 

Dysentery, tropical, entamoeba his- 
tolytica in, 328 



Ear, 383 

Earthy phosphates in urine, 87, 149 
Echinococcus disease, 348 
diagnosis, 349 
eosinophilia in, 239 
Edema, pulmonary, sputum in, 65 
Eel, vinegar, 354 

Egyptian hematuria, 167, 170, 344 
Ehrlich's diazo reaction, 126 
technic, 128 
triple stain for blood, 221 
two-cover method for blood- 
films, 217 
Einhorn's saccharimeter, 114 
Elastic fibers in sputum, 41 
Electric conductivity of urine, 79 
Elephantiasis, 356 
Embryos, filarial, in blood, 256 
filariform, 363 



Embryos of trichinella spiralis in 
blood, 257 

rhabditiform, 362 
Empty magnification, 28 
Endocarditis, malignant, vaccines 

in, 427 
Entamoeba, 328 

buccalis, 330 

coli, 329 

histolytica, 328 

in feces, 310 

tetragena, 330 
Enteritis, membranous, 313 
Enteroliths in feces, 313 
Envelope crystals in urine, 144 
Eosin and methylene-blue for blood, 

221 
Eosinophils, 239 

in sputum, 60 

in vernal catarrh, 383 
Eosinophilia, 239 

in bronchial asthma, 239 

in echinococcus disease, 239 

in filariasis, 239 

in menstruation, 239 

in myelogenous leukemia, 239, 
240 

in parasitic infections, 183 

in scarlet fever, 239, 240 

in skin diseases, 239, 240 

in trichinosis, 239 

in uncinariasis, 239 

in worm infection, 239 
Eosinophilic cells in sputum, 60 

leukocytes, 239 

in vernal catarrh, 383 

structures of blood, 221 
Epidemic cerebrospinal fever, cere- 
brospinal fluid in, 374 
Epithelial casts in urine, 158 

cells in feces, 317 
in sputum, 61 
in urine, 162, 163 
Erythroblasts, 229 
Erythrocytes, 180 

counting of, 193, 194 

enumeration of, 192 

in anemias, 226 

in gastric contents, 302 

in pernicious anemia, 226 

increase of, 192 

nucleated, 229 



INDEX 



449 



Erythrocytes, pessary forms, 226 

shape of, 226 , 

size of, 226 

stained, study of, 225 

staining properties of, variations 
in, 226 

structure, variations in, 229 

Thoma-Zeiss instrument for 
covmting, 193 
Esbach's albuminometer, 105 

estimation of proteins in milk, 
Boggs' modification, 387 

method for albumin in urine, 105 

reagent for albuminuria, 105 
Estivo-autiunnal parasite, 249, 250, 

256 
Ethereal sulphates, 90 
Ewald's salol test for gastric motor 
power, 307 

test-breakfast, 286 
Exophthalmic goiter, lymphocytes 

in, 234 
Exudates, 371 

decomposition of, indican in 
urine from, 91 
Eye, 381 
Eye-pieces, microscopic, 23 



Fahrenheit and Centigrade scales, 

440 
False albuminuria, 100 
Fasciola, 342 

hepatica, 342 
Fat in feces, 317 

in milk, estimation, 386 
Fat-droplets in sputum, 63 

in urine, 172 
Fat-globules in urine, 147 
Fatty casts in urine, 158 
Fatty-acid crystals in sputum, 42 

needles in sputum, 45 
Favus, 384 
Feces, 310 

acholic, 312 
-amebae in, 310 

animal parasites in, 313 

bacillus tuberculosis in, 319 

bacteria in, 318 
stains for, 318, 319 

bile in, 315 

bilirubin in, 315 

29 



Feces, blood in, 312, 314, 318 
blood-corpuscles in, 318 
Boas-Oppler bacillus in, 318 
calculi in, 313 
Charcot-Leyden's crystals in, 

320 
chemic examination, 314 
color, 311 
concretions in, 313 
consistence, 311 
crystals in, 319 
curds in, 314 

of milk in, 317 
enteroliths in, 313 
epithelial cells in, 317 
erj'throcytes in, 318 
examination of, chemic, 314 

macroscopic, 311 

microscopic, 315 

specimen for, 310 
fat in, 317 
flagellates in, 320 
food particles in, 316 
form, 311 

frequency of passage, 311 
functional tests, 320 
Sahli's glutoid, 321 
Schmidt's diet, 320 
gall-stones in, 313 
hydrobilirubin in, 315 
macroscopic examination, 311 
maggots in, 366 
microscopic examination, 315 
milk curds in, 317 
mucus in, 312 
muscle-fibers in, 317 
normal, 310 
occult hemorrhage in, detection, 

314 
odor, 312 
ova in, 320 
parasites in, 320 
pus in, 318 
quantity, 311 
starch granules in, 316 
tapeworms in, 313 
trypsin in, Miiller's test for, 322 
vegetable cells in, 316 

fibers in, 316 
Fehling's estimation of glucose in 

urine, 114 
test for glucose, no 



450 



INDEX 



Fermentation method of estimating 

glucose in urine, ii6 
Fibers, elastic, in sputum, 41 

in urine, extraneous, 161, 172 

muscle-, in feces, 317 

of cotton in urine, 161, 172 

of linen in urine, 161, 172 

of silk in urine, 161, 172 

of wool in urine, 161, 172 

vegetable, in feces, 316 
Fibrils, cotton, in sputum, 42 
Fibrinous casts in sputum, 45 

in urine, 157 
F"ilaria, 356 

bancrofti, 356 

diurna, 358 

infection, chyluria from, 148 

loa, 358 

medinensis, 358 

perstans, 358 

philippinensis, 358 

sanguinis hominis, 357 
Filariae in urine, 170 
Filarial embryos in blood, 256 
Filariasis, eosinophilia in, 239 

parasite of, 357 
Filariform embryos, 363 
Fish tapeworm, 351 
Fixation, chemic, for blood-films, 
219 

heat, for blood-films, 219 

of blood-films, 219 

Kowarsky's plate for, 219 
Flagellata, 327, 330 
Flagellates in feces, 320 
Flasks, 397 
Flat-worms, 340 
Flaws in slides as source of error, 

173 
Fleischl's estimation of hemoglobin, 

hemoglobinometer, 186 
Flies, 366 

Floaters in urine, 169 
Florence's reaction for detection of 
semen, 392 

reagent, 393 
Flukes, 340, 341 

liver, 342 

lung, 343 
Focal distance of objective, 23, 24 
Focusing microscope, 29 



Folin's method of quantitative 

estimation of urine, 74 
Food particles in feces, 316 

in gastric contents, 289, 302 
Formaldehyd in milk, test for, 388 
Formalin in milk, test for, 387 
Formalin-gentian- violet stain, 57 
Friinkel's diplococcus in sputum, 54 
Free acids in gastric contents, 
Congo-red test for, 290 
tests for, 289 
hydrochloric acid, 284. See also 
Hydrochloric acid, free. 
Freezing-point of urine, 79 
Friedlander's bacillus in otitis, 383 

in sputum, 58 
Frommer's test for acetone, 122 
Frothingham's method of demon- 
strating Negri bodies, 394 
modification of van Gieson's 
stain for Negri bodies, 395 
Fruit-sugar in urine, 117 
Functional tests for feces, 320 
Sahli's glutoid, 321 
Schmidt's diet, 320 
Fungi, mold, in urine, 172 
Funnel, separatory, for Strauss' 
lactic acid test, 293 



Gabbet's method for bacillus tuber- 
culosis in sputum, 49 
stain, 51 
Gall-stones in feces, 313 
Gametes in malaria, 250 
Gangrene of lung, sputum in, 65 
Gastric carcinoma, gastric contents 
in, 30s 
contents, acetic acid in, 291 
acid deficit, 299 
bacteria in, 303 
bile in, 289 
blood in, 289, 295, 302 

test for, 295 
Boas-Oppler bacillus in, 303 
butyric acid in, 291 
chemic examination, 289 
constituents, 285 
erythrocytes in, 302 
examination, 284 
chemic, 289 
microscopic, 301 



See 



Gastric contents, examination, 
physical, 288 

routine, 285 
food particles in, 289, 302 
free acids in, Congo-red test 
for, 290 
tests for, 289 
hydrochloric acid in, 284 

also Hydrochloric acid. 
in achylia gastrica, 305 
in atrophic gastritis, 305 
in carcinoma, 305 
in dilatation, 304 
in disease, 304 
in gastritis, 305 
in gastrosuccorrhea, 304 
in neuroses, 304 
in ulcer, 306 
lactic acid in, 291. See also 

Lactic acid. 
leptothrix buccalis in, 303 
microscopic examination, 301 
mucus in, 288 
obtaining, 285 
organic acids in, 291 

quantitative tests 
pepsin in, 293 

Hammerschlag's test 

Mett's test, 300 

quantitative test, 299 
Schiitz's, 300 

test for, 293 
pepsinogen in, 293 

test for, 293 
physical examination, 288 
pus-cells in, 302 
reaction, 288 

red blood-corpuscles in, 302 
rennin in, 294 

test for, 294 
renninogen in, 294 

test for, 295 
sarcinae in, 302 
tests, qualitative, 289 

quantitative, 295 
tissue bits in, 289 
total acidity, 295 
tests, 295 
Topfer's test, 295 
withdrawal, 287 
yeast-cells in, 302 
digestion, Sahli's test for, 308 



INDEX 451 

- . . . ^ ^^TV;. V 
-GfA^trfc ^uice, stimulattonj ioffifSid- 

8s' 



299 



299 



' / ; ,testrn>eals to stinlubte/ 2/^^ ^ _ 
neuroses, stomach contents m;^ / H 

ulcer, gastnc contents m, 306 ' l\ , 
Gastritis, atrophic, gastric con- 
tents in, 305 

chronic, gastric contents in, 305 
Gastro-intestinal diseases, anemia 

from, 276 
Gastrosuccorrhea, gastric contents 

in, 304 
Gauze, sterilization of, 400 
Gelatin media, sterilization of, 400 

preparation of, 402 
Gerhardt's test for diacetic acid, 

122 
Gibson's chart, 236, 237 
Giemsa's stain for syphilis, 390 
Glassware, sterilization of, 399 
Globular sputum, 67 
Glossina palpalis, 248 
Glucose in urine, 108. See also 

Glycosuria. 
Glutoid test, Sahli's, for digestive 

functions, 321 
Glycerin agar-agar, preparation of, 

402 
Glycosuria, 108 

Benedict's quantitative estima- 
tion, 115 
test in, no 

estimation of glucose, 112 

Fehling's quantitative estima- 
tion, 114 
test in, no 

fermentation method of estimat- 
ing, 116 

Haines' test in, 109 

Kowarsky's test in, in 

persistent, 109 

phenylhydrazin test in, in 

Purdy's estimation of glucose, 
112 

Robert's differential density 
method of estimating, 116 

tests, 1 09-11 6 

transitory, 108 
Gmelin's test for bile, 1 23 
Goiter, exophthalmic, lymphocytes 

in, 234 



452 



INDEX 



Goiter, lymphocytes in, 234 
Gonococcus, 41ft 

in ci>bthalmia, 382 • ■ \ ■ ■, 

, , >in pus, 36g ' 

in \irine, 169 
^ ^ v Gonorrheal ophthalmia, 382 
threads in urine, 169 
CJram-negative bacteria, 409 
Gram-positive bacteria, 409 
Gram's iodin solution, 57 

method for bacillus influenza in 
sputum, 58 
for bacteria in feces, 319 
for pus, 367 
stain for bacteria, 409 
Granular casts in urine, 157, 158 

degeneration, basophilic, 227 
Granule epithelial cells in urine, 

compound, 162 
Granules, lycopodium, in urine, 173 
starch, in feces, 316 
in urine, 173 
Gravel in urine, 142 
Grawitz's degeneration, 227 
Gray sputum, 39 
Ground itch, 361 
Guaiac test for blood, 274 

for hemoglobin, 125 
Guinea-worm, 358 
Gunning's test for acetone, 1 20 
Gutzeit's test for arsenic in urine, 
^33 



Haines' solution, no 
test for glucose, 109 

Hairs in urine, 161 

Hammerschlag's estimation of he- 
moglobin, 189 
test for pepsin, 299 

Harlow's blood stain, 224 

Hiiser's method for total solids in 
urine, 78 

Hayem, hematoblasts of, 244 

Hayem's fluid for blood count, 198 

Hay's test for bile acids, 1 24 

Heart disease, anemia from, 276 
polycythemia in, 192 

Heart-failure cells in sputum, 41. 
62, 65 

Heat and nitric acid test for al- 
bumin, 104 



I Heat fixation for blood-films, 219 
, test for albumin, Purdy's, 104 

Hematemcsis and hemoptysis, dif- 
j ferentiation, 289 

Hematoblasts of Hayem, 244 

Hematocrit, 193 
Daland's, 201 

Hematoidin crystals in sputum, 

45 
Hematuria, 166 

Egyptian, 167, 170, 344 
from kidney tubules, 166 
from pelvis of kidney, 166 
hemoglobinuria and, differentia- 
tion, 124 
idiopathic, 166 

Hemin test for blood, 274 

Hemocytometer, diluting 6uids for, 
198 
Thoma-Zeiss, 193, 194 
cleaning instrument, 199 
sources of error, 199 
technic, 195 

Hemoglobin, 184 

Dare's estimation, 189 
decrease of, 185 
estimation of, 185-191 
Hammerschlag's estimation, 189 
in urine, 124. See also Ilemo- 

globiniiria. 
medium, preparation of, 404 
Sahli's estimation, 187 
Talquist's estimation, 191 
von Fleischl's estimation, 185 

Hemoglobinometer, Dare's, 189 
Sahli's, 187, 188 
Tallquist's, 190, 191 
von Fleischl's, 186 

Hemoglobinuria, 124 
detection, 125 
guaiac test in, 125 
hematuria and, differentiation, 

124 
paroxysmal, 125 
Teichmann's test in, 125 
tests, 125 

Hemolysis, 265 

Hemolytic amboceptor, 269 

Hemoptysis and hematemesis, dif- 
ferentiation, 288 

Hemorrhage, anemia from, 276 
from bladder, 166 



INDEX 



453 



Hemorrhage from bladder, Schisto- 
somum haematobium as cause, 
167 
leukocytosis after, 207 
occult, in feces, detection, 314 
ELemosporidia, 248 
Herapathite, 137 
Herpetomonas, 335 
Hip-roof crystals in urine, 148 
Hiss' method for pneumococci in 
pus, 368 
serum media, preparation of, 405 
Hqdgkin's disease, 282 
Holt's milk-testing apparatus, 385 
Hookworm, anemia from, 276 
infection, decrease of hemoglobin 
from, 185 
diagnosis, 361 
New World, 359, 360 
Old World, 359 
Horismascope, 103 
Host, definitive, of animal para- 
sites, 324 
intermediate, of animal parasites, 

324 
Hot-air sterilizer, 396 
Human milk, 384 
Hyaline casts in urine, 154, 155 
Hydatid disease, 348 
parasite of, 349 
Hydrobilirubin in feces, 315 
Hydrochloric acid, combined, 284 
Topfer's test for, 298 
free, 284 
absence, 297 

amidobenzol test for, 290 
amount, 297 
decrease, 297 
Boas' test for, 290 
increase, 297 
tests for, 290 

quantitative, 296 
Topfer's test for, 297 
Hydrogen sulphid generator, 135 
Hydrophobia, 393. See also 

- Rabies. 
HjTTienolepis, 350 

nana, 350 
HjTDerchlorhydria, 297 
H^-perchromemia, 184 
Hyperemia, active, urine in, 173 
passive, urine in, 174 



Hyperemia, renal, urine in, 173 
Hyphae of molds in urine, 161 
Hypobromite method for urea in 

urine, 94 
Hypochlorhydria, 297 
Hypodermic injection of tubercu- 

Im for diagnosis, 429 



Idiopathic hematuria, 166 

polycythemia, 185, 192 
Illumination, dark ground, of mi- 
croscope, 21 
for microscope, 18 

with water-bottle condenser, 19 
Immersion objective, 24 
Immune bodies, 265, 266 
Incidental parasites, 340 
Incubation of bacteria, 413 
Incubator, 397 
Index, color, of blood, 200 
opsonic, 263 
phagocytic, 263 
volume, of blood, 200 

Larrabee's method, 201 
method, 201 
India-ink method for syphilis, 391 
Indican in urine, 91 
detection, 91 

from decomposition of exu- 
dates, 91 
from diminished flow of bile, 91 
in biliousness, 90 
in diseases of small intestine, 90 

of stomach, 90 
Obermayer's test for, 91 
tests for, 91 
Indicanuria, 90. See also Indican 

in urine. 
Infection, blood-plaques in, 214 
leukocytosis in, 206 
mixed, 54 

phagocytosis and, 261 
vaccines in, 426, 427 
Infecrious diseases, secondary ane- 
mia from, 276 
Inflammations, leukocytosis in, 206 
pseudomembranous, of mouth, 
378 
Influenza bacillus, 418 
in spinal fluid, 376 
in sputum, 58 



454 



INDEX 



Infusion, beef, preparation of, 401 
bouillon, preparation of, 401 

Infusoria, 328, 339 

Inoculating media for bacteria, 412 

Inoculation, animal, 375 
of bacteria, 415 

Intermediate host of animal para- 
sites, 324 

Intestine, small, diseases of, indican 
in urine in, 90 

Intoxication, acid, cause, 118 

lodin in urine, 133 

reaction of leukocytes, 238 
solution. Gram's, 57 

Iodoform crystals from Gunning's 
test, 121 

lodophilia, 238 

Irregular malaria, 249 

Itch, ground, 361 
mite, 366 



Kala-azar, Leishmania donovani 

of, 335 

Kelling's test for lactic acid, 292 

Kemp-Calhoun-Harris estimation 
of blood-plaques, 214 

Kidney, changes in, albuminuria 
from, loi 
permeability of, tests for, 78, 79 

Koch-Weeks bacillus in conjuncti- 
vitis, 381 

Kowarsky's plate for fixation of 
blood-films, 219 
test for glucose, 11 1 



Lactic acid in gastric contents, 291 
Kelling's test for, 292 
Simon's test for, 292 
Strauss' test for, 292 
Uffelmann's test for, 292 
Lactose in milk, estimation, 387 

in urine, 117 
Lamblia, 338 

intestinalis, 338 
Lamp, Matthews' microscope, 19, 

20 
Lancet, blood-, 183 
Lange's test for acetone, 121 
Larrabee's estimation of volume 
index of blood, 201 



Lead in urine, 134 

Lederer's test, 134 
Lead-poisoning, anemia from, 276 

chronic, degeneration of Grawitz 
in, 228 
Lederer's test for lead in urine, 134 
Lefifmann-Beam estimation of fat 

in milk, 386 
Legal 's test for acetone, Lange's 

modification, 121 
Leishman-Donovan bodies, 335 
Leishmania, 335 

donovani, 335 

infantum, 335 

tropica, 335 
Leishman's method for measuring 

opsonins, 263 
Lenses, 23 

for microscope, care of, 30 
Leprosy, secondary anemia from, 

276 
Leptothrix buccalis, 377 
in gastric contents, 303 
in sputum, 42 
Leucin in urine, 145 
Leukemia, 203, 208, 280 

blood-plaques in, 214 

Boggs' estimation of leukocytes 
in, 209 

degeneration of Grawitz in, 228 

diluting fluids for count, 213 
Leukemia, eosinophilia in, 239, 240 

erythroblasts in, 230 

leukocyte count in, 209 

lymphatic, 281 

lymphocytes in, 234 

mast-cells in, 241 

myelocytes in, 242 

myelogenous, 280 

oligocythemia in, 193 

polychromatophilia in, 227 

Todd's estimation of leukocytes 
in, 211 

Turck's ruling for blood count in, 
209, 211 

Zappert ruling for blood count 
in, 209 
Leukocytes, 180 

abnormal varieties, 241 

atypic forms, 242 

basophilic, 240 

border-line forms, 243 



INDEX 



455 



Leukocytes, classification of, 231 
counting, in leukemia, 209 
decrease in, 201 
degenerated forms, 243 
differential count of, 230 
enumeration, 202 
eosinophilic, 239. See also 

Eosinopkiles. 
increase in, 203 

absolute, 231 

relative, 231 
iodin reaction of, 238 
irritation forms, 243 
mononuclear, large, 234 
normal, 232 
polymorphonuclear, neutrophilic, 

235 
polynuclear, 236 
stained, study of, 230 
transitional, 235 
vacuolated, 243 
Leukocytosis, 203 
absolute, 231 
digestive, 205 
lymphocyte, 204, 207 

in hereditary syphilis, 208 
in pertussis, 208 
myelocytes in, 242 
non-phagocytic, 208 
permanent, 203 
polymorphonuclear, 204 
from drugs, 207 
from infections, 206 
from inflammations, 206 
in malignant disease, 207 
pathologic, 205 
physiologic, 205 
toxic, 207 
relative, 231 
transient, 203 
Leukopenia, 202 
in chlorosis, 202 
in pernicious anemia, 202 
lymphocytes in, 233 
Levulose in urine, 117 
Lieben's test for acetone. Gunning's 

modification, 121 
Linen fibers in urine, 161, 172 
Litmus milk, preparation of, 404 
Liver, cirrhosis of, anemia from, 276 
fluke, 342 
rot, 342 



Loffler's alkaline methylene-blue, 

57 
blood-senmi, preparation of, 403 
methylene-blue for gonococci in 
pus, 369 
for pus, 367 
stain for flagella, 411 
Louse, 366 

Lung, edema of, sputum in, 65 
fluke, 343 

gangrene of, sputum in, 65 
tuberculosis of, sputiun in, 66 
Lycopodium granules in urine, 173 

used as micrometer, 33 
LjTnphatic leukemia, 281 
Ijrmphocytes in, 234 
Lymphocyte leukocytosis, 204, 207 
in hereditary syphilis, 208 
in pertussis, 208 
Lymphocytes, 232 
Lymphocytosis, 208 



Macrocytes, 226 

Macroscopic examination of spu- 
tum, 37 

Maggots in feces, 366 

Magnification, empty, 28 
microscopic, 27 
methods of increasing, 28 

Malaria, irregular, 249 
large mononuclear leukocytes in, 

234 
parasites of, 248. See also 

Malarial parasites. 
secondary anemia from, 276 
transmission of, by mosquitos, 

250 
Malarial parasites, 248 

ameboid movements of, 248 

cycles of, 248 

asexual cycle, 248 

detection, 251, 253, 254 

estivo-autumnal, 249, 250, 256 

gametes in blood with, 250 

hyaline stage of, 248 

life histories, 248 

merozoites of, 248 

mosquitos as host, 250 

quartan, 250, 256 

Ruge's stain for, 254 

segmentation of, 248 



456 



INDEX 



Malarial parasites, sexual cycle, 
248, 249 
spores of, 248 
stains for, 254 
tertian, 249, 250, 256 
Wright's stain for, 254 
stippling, 228 
Malignant disease, leukocytosis in, 
207 
endocarditis, vaccines in, 427 
tumors, anemia from, 276 
Mast-cells, 240 
Mastigophora, 327, 330 
Matthews' microscope lamp, 19. 20 
McFarland's method for Widal 

reaction, 259 
Measles, diazo reaction in, 128 
Measures, 439 

Media, culture-, 401. See also 
Culture-media. 
gelatin, sterilization of, 400 
Megaloblasts, 229 
Megalocytes, 226 
Melanin in urine, 126 

tests for, 126 
Melanogen in urine, 126 
^lelanuria, 126 
Membranous enteritis, 313 
Meningitis, tuberculous, cerebro- 
spinal fluid in, 374 
Menstruation, eosinophilia during, 

239 
Mercury in urine, 136 

treatment of syphilis, effect of, 
on Wassermann reaction, 273 
Merozoites of malarial parasites. 

248 
Metal, sterilization of, 399 
Methylene-blue eosin stains, poly- 
chrome, for blood, 222 

test for urine, 79 
Metric system, 439 
Mett's test for pepsin, 300 

tubes, 399 
Microblasts, 229 

Micrococcus catarrhalis in sputum, 
58. 59 

urae in urine, 167 
Microcytes, 226 

Micrometer eye-piece for micro- 
scope, 31, 32 

stage, 32 



Micron, 32 
Microscope, 17 

care of, 30 

choice of, 31 

cleaning, 30 

condenser for, 22 

eye-pieces for, 23 

focusing, 28 

illumination for, 18 
dark ground, 21 

lamp, Aiatthews, 19, 20 

lenses for, 23 
care of, 30 

magnification by, 27 

methods of increasing, 28 

method of carrying, 30 

micrometer eye-piece for, 31, 32 

objectives for, 23 
corrections, 2*4 

use, 17 
^licroscopic objects, measurement, 

31 

Micturition, frequency of, 70 

Milk, 384 

analysis of, 384 
tube for, 386 
bacteria in, 384 
chemic examination, 385 
curds of, in feces, 317 
fat in, estimation, 386 
formalin in, test for, 387 
lactose in, estimation, 387 
litmus, preparation of, 404 
proteins in, estimation, 387 
reaction, 384 

Milk-sugar in urine, 117 

Milk-testing apparatus. Holt's, 385 

Mineral sulphates, 90 

Mite, itch, 366 

Mixed infection, 54 

Moeller's stain for spores, 410 

Mold fungi in urine, 172 

Molds, hj-pha; of, in urine, 161 
in sputum, 47 

Mononuclear leukoc>'tes, large, 234 

Morax and Axenfeld's diplobacil- 
lus, 381 

Moro's tuberculin reaction in diag- 
nosis of tuberculosis, 430 

Morphin in urine, 136 

Mosquitos in transmission of mala- 
ria, 250 



INDEX 



457 



Motor power of stomach, 307 
Mouth, diseases of, 377 

organism of, 377 
Mucin in urine, 106 
Mucous threads in urine, 159 
Mucus in feces, 312 
in gastric contents, 288 
in urine, 159 
Miiller's blood-dust, 181 
fluid, 56 

test for trypsin in feces, 322 
Muscle-fibers in feces, 317 
Myelin globules in sputum, 63 
Myelocytes, 241 
Myelogenous leukemia, 280 
eosinophilia in, 239, 240 
erythroblasts in, 230 
mast-cells in, 241 
myelocytes in, 242 

Nagana, trypanosoma brucei of, 

335 
Nasopharynx, diphtheria of, 378 
Necator americanus, 359, 360 

life-history, 360 
Needles, fatty-acid, in sputum, 45 
Negative-staining of urinary casts, 

154 
Negri bodies, 393 

Frothingham's method of de- 
monstrating, 394 
Van Gieson's stain for, Froth- 
ingham's modification, 395 
Nemathelminthes, 341, 353 
Nematoda, 353 
Nematodes, 353 
Nephritis, anemia from, 276 

chlorids in urine in, 82 

urine in, 173, 175, 176 
Neuroses, gastric, stomach contents 

in, 304 
Neutrophilic leukocytes, polymor- 
phonuclear, 235 

structures of blood, 221 
Newton's rings, 196 
Nitric acid test for albumin, 104 
Nitrogen equilibrium, 92 

partition, 92 
Noguchi test for syphilis, 271 
Normoblasts, 229 
Nose, cylindric cells from, in spu- 

timi, 6i 



Nubecula of urine, 72 
Numeric aperture, 25 
Nutrition, poor, secondary anemia 
from, 276 



Obermayer's reagent, 91 

test for indican in urine, 91 
Objectives, achromatic, 23 

apochromatic, 23 

dry, 24 

focal distance of, 23, 24 

immersion, 24 

microscopic, 23 

niuneric apertures, 25 

oil-immersion, 24, 25 

resolving power of, 25 

working distance of, 23 
Oblique illumination of microscope, 

20 
Occult hemorrhage in feces, detec- 
tion, 314 
O'idlum albicans, 378 
Oil-immersion objective, 24, 25 
Oligochromemia, 185 
Oligocythemia, 192 

in anemias, 193 

in chlorosis, 193 

in leukemia, 193 
Oliguria, 71 
Oncospheres, 346 
Ophthalmia, gonorrheal, 382 
Ophthalmo-tuberculin reaction, 

Calmette's, 430 
Opisthorchis, 343 

felineus, 343 

sinensis, 343 
Opsonic index, 263 
Opsonins, 261 

Leishman's method for measur- 
ing, 263 

measuring amount of, 262 

Wright's method for measuring, 
262 
Orcin test, Bial's, for pentoses, 118 
Organic acids in gastric contents, 
291 
quantitative tests, 299 
Oriental sore, Leishmania tropica 

of, 235 
Orthostatic albuminuria, 100 
Otitis, 383 



458 



INDEX 



Otitis, bacteria of, 383 

tuberculous, 383 
Ova in feces, 320 
Oxybutyric acid in urine, 123 
Oxj^ihilic structures of blood, 221 
Oxyuris, 355 

vermicularis, 355 
ova of, 356 



Pancreatic reaction, 129 

flasks for, 130 

in pancreatitis, 129 

technic, 129 
Pancreatitis, pancreatic reaction in, 

129 
Pappenheim's method for bacillus 

tuberculosis in sputum, 51 
pyronin-methyl-green for bac- 
teria, 408 

for gonococci in pus, 369 

for pus, 367 
Paragonimus, 343 
westermani, 343 

in sputum, 48 
Paramoecium coli, 339 
Parasites, animal, 323 

anemia from, 276 

arthroiX)da, 366 

classification, 324, 325 

definitive host, 324 

in blood, 247 

in feces, 313 

in sputum, 48 

in urine, 169 

infection with, eosinophilia in, 

239 
intermediate host, 324 
nomenclature, 324, 325 
protozoa, 326, 327 
blood, 244 

causing skin diseases, 384 
in feces, 320 
incidental, 340 

malarial, 248. See also Malarial 
parasites. 
Paroxysmal hemoglobinuria, 125 
Pavement epithelial cells in urine, 

163 
Pediculus capitis, 366 
pubis, 366 
vestimenti, 366 



Pemphigus, eosinophilia in, 240 
Pentoses in urine, 117 

Bial's orcin test, 118 
Pepsin in gastric contents, 293 

Hammerschlag's test, 299 
Mett's test, 300 
quantitative test, 299 
Schutz's law, 300 
test for, 293 
Pepsinogen in gastric contents, 293 

test for, 293 
Peptone solution, Dunham's, prep- 
aration of, 404 
Pericardial fluid, examination, 371 
Peritoneal fluid, examination, 371 
Permeability of kidneys, test, 78, 79 
Pernicious anemia, 277 
blood-plaques in, 214 
color index in, 200 
degeneration of Grawitz in, 228 
erythroblasts in blood in, 230 
erythrocytes in, 226 
leukopenia in, 202 
lymphocytes in, 234 
myelocytes in, 242 
polychromatophilia in, 227 
Pertussis, lymphocyte leukocytosis 
in, 208 
lymphocytes in, 234 
Pessary forms of erythrocytes, 226 
Pfeiffer's phenomenon, 265 
Phagocytic index, 263 
Phagocytosis, 206 

and infection, 261 
Pharyngomycosis leptothrica, 377 
Pharynx, tuberculosis of, 380 

ulceration of, 380 
Phenacetin in urine, 132 
Phenol in urine, 136 
Phenolphthalein in urine, 137. 
Phenylglucosazone crystals, no 
Phenylhydrazin test for glucose, 

III 
Phloridzin test for urine, 79 . 
Phosphate crystals in urine, am- 
moniomagnesium, 148 
calcium, 149 
triple, 148 
Phosphates in urine, 86, 148 
alkaline, 87 

amorphous, 72, 87, 149 
in mass, 160 



INDEX 



459 



Phosphates in urine, decreased, 87 
earthy, 87, 149 
estimation, 87, 88 
Purdy's centrifuge method, 

88 
quantitative, 87 
increased, 87 
Purdy's table for, after cen- 

trifugation, 88 
triple, 87 
Phosphaturia, 87 
Phosphorus-poisoning, anemia 

from, 276 
Photomicrography, S3 
Physiologic albuminuria, 100 
Pink-eye, 381 
Pin- worm, 355 
Pipets, 398 

for counting vaccines by Wright's 
method, 422 
Piroplasma hominis, 339 
Pirquet's reaction in tuberculosis, 

430 
Plasmodium, 338, 339 

falciparum, 248 

malariae, 248. See also Malarial 
parasites. 

vivax, 248 
Platinum wires, 397 
Platyhelminthes, 340, 341 
Pleural fluid, examination, 371 
Plugs, Dittrich's, in sputum, 39 
Pneumococcus, 416 

capsules, Buerger's method for, 

55 
in eye affections, 381 

otitis, 383 
in pus, 368 
sputum, 54 
Smith's method, 56 
Pneumonia, croupous, sputum in, 
66 
drunkard's, sputum in, 39 
Poikilocytes, 226 
Poikilocytosis, 226 
Poisoning, arsenic, anemia from, 
276 
lead-, anemia from, 276 
phosphorus-, anemia from, 276 
Polychromatophilia , 227 
Polychrome methylene-blue eosin 
stains for blood, 222 



Polycythemia, 192 

idiopathic, 185, 192 

in diarrhea, 192 

in heart disease, 192 
Polyhedral cells in urine, 162 
Polymorphonuclear leukocytosis, 

204, 205. See also Leukocytosis, 
polymorphonuclear. 

neutrophilic leukocytes, 235 
Polynuclear leukocytes, 236 
Polyuria, 70 
Pork tapeworm, 348 
Posthemorrhagic leukocytosis, 207 
Postural albuminuria, 100 
Potassium indoxyl sulphate in 

urine, 90. See also Indican in 

urine. 
Potato medium, preparation of, 404 
Power of resistance, 236 
Preformed sulphates, 90 
Pregnancy, urine in, 175 
Primary proteoses in urine, 106 
Proglottides, 345 

Progressive pernicious anemia, 277 
Proteins in milk, estimation, 387 

in urine, 99 
Proteoses in urine, 106 
detection, 108 
primary, 106 
secondary, 106 
Protozoa, 326, 327 
Prune-juice sputum, 39 
Prurigo, eosinophilia in, 240 
Pseudocasts in urine, 161 
Pseudoleukemia, 282 
Pseudomembranous conjunctivits, 
382 

inflammations of mouth, 378 
Psoriasis, eosinophilia in, 240 
Pulmonary edema, sputum in, 65 

gangrene, sputum in, 65 

tuberculosis, sputum in, 66 
tuberculin in, 428, 249 
Purdy's centrifugal estimation of 
albumin, 106 
of chlorids, 83 
of phosphates, 88 
of sulphates, 89 

centrifuge tubes, 85 

electric centrifuge, 83 

estimation of glucose in urine, 112 

heat test for albumin, 104 



460 



INDEX 



Purdy's solution for glucose test, 

table for estimation of albumin, 
107 
of chlorids, 86 
of phosphates, 88 
of sulphates, 89 
Purin bodies in urine, 95 

Cook's method, 96 
Purpura haemorrhagica, blood- 
plaques in, 214 
Pus, bacillus tuberculosis in, 369 
bacteria in, 367 
examination of. 367 
gonococci in, 369 
Gram's method for, 367 
in feces, 318 
in urine, 72, 164 

Donne's test, 72 
Lofller's methylene-blue for, 367 
Pappenheim's pyronin-methyl- 

green for, 367 
pneumococci in, 368 
staphylococci in, 368 
streptococci in, 368 
Pus-casts in urine, 159 
Pus-corpuscles, 236, 367 
in feces, 318 
in gastric contents, 302 
in sputum, 59 
in urine, 163 
Pyelitis, urine in, 177 
Pyuria, 164 



Quartan parasite, 250, 256 
Quinin in urine, 137 



Rabies, diagnosis of, 393 

Frothingham's method of de- 
monstrating Negri bodies in, 

3Q4 
Ray-fungus in sputum, 46 
Reaction, Noguchi's, for syphilis, 

271 
Wassermann, 274. See also 

Wasserniann reaction. 
Reagents. 434 
Red blocxi-corpuscles, 180 

decrease of, 192. See also 
Oligocythemia. 



Red blood-corpuscles in gastric 
contents, 302 
in sputum, 63 
in urine, 165 

increase of, 192. See also 
Polycytticmia. 
sand in urine, 142 
Reinsch's test for arsenic in urine, 

133 
Relapsing fever, spirochaeta of, 247, 

331 
Renal albuminuria, 100 

calculus, urine in, 177 

circulation, changes in, albu- 
minuria from, loi 

hyperemia, urine in, 173 

tuberculosis, urine in, 175 
Rennin in gastric contents, 294 

test for, 294 
Renninogcn in gastric contents, 294 

test for, 295 
Resinous drugs in urine, 137 
Resistance, power of, 236 
Resolving jx)\ver of objective, 25 
Rhabditiform embryos, 362 
Rheumatism, secondary anemia 

from, 276 
Rhizojxxla, 327, 328 
Rice's solutions, 95 
Ring bodies, Cabot's, 230 
Rings, Xewton's, 196 
Ringworm. 384 

Robert's differential density meth- 
od of estimating glucose in 
urine, 116 

test for albumin, 103 
Ronchese-Malfatti formalin test for 

ammonia in urine, 98 
Round- worms, 354 

in children, 354 
Ruge's stain for malarial parasites, 

254 
Ruhemann's method for uric add, 

97 
reagent, 97 
uricometer, 96 
Rusty sputum, 38, 39 



Saccharimeter, Einhom's, 1 14 
Sahli's desmoid test of gastric di- 
gestion, 308 



INDEX 



461 



Sahli's estimation of hemoglobin, 
187 

glutoid test for digestive func- 
tions, 321 

hemoglobinometer, 187, 188 
Salicylates in urine, 137 
Salol in urine, 137 

test, Ewald's, for gastric motor 
power, 307 
Salvarsan treatment of syphilis, 

effect of, on Wassermann reac- 
tion, 273 
Sand, red, in urine, 142 
Sarcinae in gastric contents, 302 
Sarcodina, 327, 328 
Sarcoptes scabiei, 366 
Saxe's urinopyknometer, 76 
Scarlet fever, eosinophilia in, 239, 

240 
Schistosomum, 344 

hematobium, 342, 344 
in urine, 170 

in veins of bladder as cause of 
hemorrhage, 167 

japonicum, 345 
Schmidt's diet for examination of 

feces, 320 
Schiitz's law in quantitative test for 

pepsin, 300 
Scolex, 345 
Scratches on slide as source of 

error, 173 
Screw worm, 366 
Secondary anemia, 276 

proteoses in urine, 106 
Secretory ability of kidneys, tests, 

78, 79 
Sediments, urinary, 138. See also 

Urinary sediment. 
Segmentation of malarial parasites, 

248 
Semen, examination of, 391 

on clothes, detection, 392 
Florence's reaction for, 392 
Separatory funnel for Strauss' 

lactic acid test, 293 
Scrum, blood-, Loffler's, prepara- 
tion of, 403 

media, Hiss', preparation of, 405 

reactions, 257 
Serum-albumin in urine, 99 
Serum-globulin in urine, 99 



Shadow cells in urine, 166 
Silk fibers in urine, 161, 172 
Silver impregnation method for 
syphilis, 390 

nitrate solution, ammoniated, 97 
Simon's test for lactic acid, 292 
Skin diseases, eosinophilia in, 239, 
240 

parasitic diseases of, 384 
Sleeping sickness, 248 

trypanosoma gambiense of, 334 
Small intestine, diseases of, indican 

in urine in, 90 
Smegma bacillus, 53, 169 
Smith's method for pneumococcus 
in sputum, 56 

test for bile, 123 
Sodium urate in urine, 144 
Specific gravity of urine, 74 
Spermatozoa, absence of, 391 

in urine, 167 
Spinal fluid, influenza bacilli in, 376 
Spirals, Curschmann's, in sputum, 

43 
Spirochaeta, 330 

buccalis, 332 

carter i, 331 

dentium, 332 

duttoni, 331 

kochi, 331 

novyi, 331 

obermeieri, 331 

pallida, 388 

recurrentis, 330 
in blood, 247 

refringens, 2>3,3 
in syphilis, 389 

vincenti, 331, 380 
Splenic anemia, 280 
Splenomegaly, infantile, Leish- 

mania infantum of, 335 
Spores of malarial parasites, 248 
Sporozoa, 328, 338 
Sputum, 36 

actinomyces bovis in, 46 

albumin in, 63 

alveolar cells in, 62 

Amceba histolytica in, 48 

animal parasites in, 48 

bacillus mucosus capsulatus in, 
58 
of Friedlander in, 58 



462 



INDEX 



Sputum, bacillus of influenza in, 58 
tuberculosis in, 36, 49 

bacteria in, 49 

black, 39 

cells in, 59 
stains for, 59 

Charcot-Leyden crystals in, 44, 

45 
chemic examination, 63 
cholesterin crystals in, 45 
ciliated body-cells in, 48 
collection of, 36, 37 
color of, 38 
consistence, 39 
cotton fibrils in, 42 
crudum, 39 
crystals in, 44, 45 
Curschmann's spirals in, 43 
cylindric cells in, 61 
diplococcus of Frankel in, 54 
Dittrich's plugs in, 39 
elastic fibers in, 41 
eosinophilic cells in, 60 
epithelial cells in, 61 
examination, 36 

chemic, 63 

macroscopic, 37 

microscopic, 40 

physical, 38 
fat-droplets in, 63 
fatty-acid crystals in, 42 

needles in, 45 
fibrinous casts in, 45 
Frankel's diplococcus in, 54 
globular, 67 
gray, 39 

heart-failure cells in, 41, 62, 65 
hematoidin crystals in, 45 
in bronchial asthma, 66 
in bronchiectasis, 65 
in bronchitis, 64 
in croupous pneumonia, 66 
in disease, 64 

in drunkard's pneumonia. 39 
in gangrene of lung, 65 
in pneumonia, croupous, 66 
in pulmonary edema, 65 

gangrene, 65 

tuberculosis, 66 
leptothrix buccalis in, 42 
macroscopic examination, 37 
micrococcus catarrhalis in, 58, 59 



Sputum, molds in, 47 

myelin globules in, 63 

Paragonimus westermani in, 48 

pneumococcus in, 54 
Smith's method, 56 

prune-juice, 39 

pus-corpuscles in, 59 

quantity, 38 

ray-fungus in, 46 

receptacle for, 37 

rusty, 38, 39 

squamous cells in, 61 

stained, 48 

staphylococci in, 54 

streptococci in, 54 

streptothrix actinomyces in, 47 

tubercle bacillus in, 36, 49 

unstained, 40 

yeasts in, 47 
Squamous cells in sputum, 61 

epithelial cells in urine, 163 
Squibb's urinometer, 75 
Stage micrometer, 32 
Stained blood, 216 

sputum, 48 
Staining methods, 407 
Stains, 434 

anilin, for blood- films, 220 

anilin-gentian violet, 57 

carbol fuchsin, 50 

thionin, for bacteria, 408 

Ehrlich's triple, for blood, 221 

eosin and methylene-blue, for 
blood, 221 

for bacillus influenza in sputum, 

tuberculosis in sputum, 49 
for bacteria, 407 

in feces, 318 

in sputum, 49 

for morphology, 407 
for blood, 216 

Wright's, in cytodiagnosis, 372 
for blood-films, 216, 220 
for cells in sputum, 59 
for malarial parasites, 254 
for pneumococcus capsules, 55 
for pus, 367 
for syphilis, 390 
formalin-gentian-violet, 57 
Gabbet's, 51 
Giemsa's, for syphilis, 390 



INDEX 



463 



Stains, Gram's, for bacteria, 409 

for pus, 367 

iodin solution, 57 
Harlow's, for blood, 224 
India-ink, for syphilis, 391 
iodin, for leukocytes, 238 
iodin solution. Gram's, 57 
LoflBer's alkaline methylene-blue, 

57 
for flagella, 411 

methylene-blue, for gonococci 
in pus, 369 
for pus, 367 
Moeller's, for spores, 410 
negative-, for urinary casts, 154 
Pappenheim's pyronin-methyl- 
green, 367 
for bacteria, 408 
for gonococci in pus, 369 
polychrome methylene-blue eosin , 

for blood, 222 
Ruge's, for malarial parasites, 254 
silver, for syphilis, 390 
Stirling's anilin-gentian-violet, 

57 
triple, for blood, 221 
Van Gieson's, for Negri bodies, 

Frothingham's modification, 

395 
Wright's, for blood, 222 
in cytodiagnosis, 372 
for malarial parasites, 254 
for syphilis, 390 
Staphylococci, 368 
in eye affections, 381 
in otitis, 383 
in sputum, 54 
Staphylococcus pyogenes albus, 416 
aureus, 415 
citreus, 416 
Starch paper, 307 
Starch-granules in feces, 316 

in urine, 173 
Steam sterilizer, 396 
Sterility, 391 
Sterilization, 399 

in preparation of vaccines, 422 

of cotton, 400 

of culture-media, 399 

of gauze, 400 

of gelatin media, 400 

of glassware, 399 



Sterilization of metal, 399 
Sterilizers, 396 

dry, 396 

hot-air, 396 

steam, 396 
Stippling, malarial, 228 
Stirling's anilin-gentian-violet stain, 

57 
Stock vaccines, 419 
Stomach, 284 

absorptive power of, 306 

contents of, 284. See also Gastric 
contents. 

digestion, 284 

dilatation of, gastric contents in, 

.304 
diseases of, indican in urine in, 90 
motor power of, 307 
position of, determination, 308 
size of, determination, 308 
Stomach- tube, 287, 288 
Stools, 310. See also Feces. 
Strauss' test for lactic acid, 292 
Streptococci, 368 
in eye affections, 381, 382 
in otitis, 383 
in sputum, 54 
Streptococcus pyogenes, 416 
Streptothrix actinomyces in spu- 
tum, 47 
Strongyloides, 362 

intestinalis, 324, 362, 363 
Sugar media, preparation of, 403 
Sugars in urine, 108 
Sulphates, conjugate, 90 
ethereal, 90 
in urine, 88 
estimation, Purdy's centrifugal 
method, 89 
quantitative, 89 
Purdy's table after centrifuga- 
tion, 89 
mineral, 90 
preformed, 90 
Sulphuric acid in urine, 88 
Surra, trypanosoma evansi of, 335 
Syphilis, dark ground illumination 
in, 391 
examination of material, 388 
Giemsa's stain for, 390 
hereditary, lymphocyte leuko- 
cytosis in, 208 



464 



INDEX 



Syphilis, India-ink method for, 391 
micro-organism of, 388, 389 
Noguchi reaction for, 271 
secondary anemia from, 276 
silver impreganation method for, 

390 
spirochajta pallida in, 388 

refringens in, 389 
treponema pallidum in, ^i^^, 388, 
389 
stains for, 390 
Wassermann reaction for, 264. 
See also Wassrrniann reaction. 
Wright's stain for, 390 



T.KNiA, 347 

echinococcus, 346, 348, 349 
in urine, 169 

elliptica, 351 

mediocanellata, 347 

saginata, 345, 346, 347 

solium, 345, 346, 346 
Tallquist's estimation of hemo- 
globin, 191 

hemoglobinometer, 190, 191 
Tannin in urine, 138 
Tapeworm, 341, 345 

beef, 347 

dwarf, 350 

fish, 351 

in feces, 313 

pork, 348 
Teichmann's test for blood, 274 

for hemoglobinuria, 125 
Telosporidia, 328, 338 
Temperature, 440 
Tertian parasite, 249, 250, 256 
Test-breakfast, Boas', 286 

Ewald's, 286 
Test-meals, 286 

Boas', 286 

Ewald's, 286 
Texas fever, Babesia bigeminum of, 

339 
Thoma-Zeiss hemocytometer, 193, 
194 
cleaning iru^trument, 199 
sources of error, 199 
technic, 195 
Thorn-apple crystals in urine, 151 
Thread- worm, 355 



Thrush, 378 

Tick fever, Babesia hominis of, 339 

Tinea versicolor, 384 

Tissue bits in gastric contents, 289 

Todd's estimation of leukocytes in 

leukemia, 211 
Toisson's fluid for blood count, 

198 
Tonsils, diphtheria of, 378 
Tdpfcr's test for combined hydro- 
chloric acid, 298 
for free hydrochloric acid, 297 
for total acidity, 295 
Torfuge, Wethcrill's, 139 
T. O. tuberculin, 428 
Toxic absorption, degree of, 236 

leukocytosis, 207 
T. R. tuberculin, 428 
Trachea, cylindric cells from, in 

sputum, 61 
Trachoma, 383 
Transitional leukocytes, 235 
Transitory glycosuria, 108 
Transudates, 371 
Trematoda, 340, 341 
Trematodes, 340, 341 
Treponema, ;i$^ 

pallidum, 333, 388, 389 
Giemsa's stain for, 390 
India-ink method, 391 
silver impregnation method, 

390 
Wright's stain for, 390 
pertenue, 333 
Trichinella, 363 

spiralis, 324, 363, 364 
embryos of, in blood, 257 
Trichiniasis, diagnosis, 364 

parasite of, 363 
Trichinosis, eosinophilia in, 239 
Trichloracetic acid test for albumin, 

102 
Trichocephalus, 364 

trichiurus, 364 
Trichomonas, 336 
intestinalis, 337 
pulmonalis, 337 
vaginalis, 336 
in urine, 170 
Triple phosphate crystals in urine, 
148 
phosphates in urine, 87 



INDEX 



465 



Triple stain, Ehrlich's, for blood, 

221 
Tropical dysentery, entamoeba his- 
tolytica in, 328 
Trypanosoma, SSS 

brucei, 335 

cruzi, 334 

equiperdum, 335 

evansi, 335 

gambiensi, 334 
in blood, 247 

lewisi, 334 
Trypanosomes, 333 

blepharoplast of, ^3$ 
Trypsin in feces, Miiller's test for, 

322 
Tsuchiya's method for albumin in 

urine, 105 
Tube-casts in urine, 152 
Tubercle bacillus. See also Bacil- 
lus tuberculosis. 
Tuberculin, 428 

B. E., 428 

B. F., 428 

dosage of, 428 

in diagnosis, 429 

Calmette's oph thai mo- tuber- 
culin reaction, 430 
hypodermic injection, 429 
Moro's reaction, 430 
Von Pirquet's reaction, 430 

in tuberculosis, 428 

reaction of, 429 

T. O., 428 

T. R., 428 
Tuberculosis, animal inoculation 
in, 375 

Calmette's reaction in, 430 

diazo reaction in, 127 

Moro's reaction in, 430 

of mouth, 380 

of pharynx, 380 

pulmonary, sputum in, 66 

renal, urine in, 175 

secondary anemia from, 276 

tuberculin in diagnosis of, 429 
in treatment of, 428 

vesical, urine in, 178 

Von Pirquet's reaction in, 430 
Tubing culture-media, 406 
Tumors, malignant, anemia from, 

276 

30 



Tumors, vesical, urine in, 178 
Tiirck's ruling for blood count in 

leukemia, 209, 211 
Turpentine, odor of urine from, 73 
Two-slide method for blood-films, 

217 
Typhoid bacilllus, 417 
in blood, 244 
technic, 245 
fever, diazo reaction in, 127 
lymphocytes in, 234 
secondary anemia from, 276 
vaccines in, 428 
Widal reaction in, 127, 258 
Tyrosin in urine, 145, 146 



Uffelmann's test for lactic acid, 

292 
Ulcer, gastric, stomach contents in, 

306 
Ulcerations of mouth, 380 

of pharynx, 380 
Uncinaria duodenalis, 359 

life-history, 360 
Uncinariasis, anemia from, 276 

diagnosis of, 361 

eosinophilia in, 239 
Unstained sputum, 40 
Urates, amorphous, in urine, 72, 95, 

143 
in mass, 160 
Urea in urine, 92 
decreased, 93 

estimation, quantitative, 94 
increased, 92 
tests, 94 
Ureometer, Doremus-Hinds', 94 
Uric acid crj'stals in urine, 142 
in urine, 95 

Cook's method, 96 
decreased, 96 

estimation, quantitative, 96 
increased, 96 
Ruhemann's method, 97 
Uricometer, Ruhemann's, 96 
Urinary albumin, 99 
crystals, 141, 142 
sediment, examination, 138 
organized, 151 
transference to slide, 138 
imorganized, 141 



466 



INDEX 



Urinary sediment, unorganized, 
in acid urine, 141, 142 
in alkaline urine, 142, 148 
Urine, 68 

acctanilid in, 132 

acetone in, 118. See also 

Acelonuria. 
acid, 73 

unorganized sediments in, 141, 
.142 
acidity, quantitative estimation, 

74 
P'olin's method, 74 

air bubbles in, 173 

albumin in, 99. See also Albu- 
minuria. 

alkaline, unorganized sediments 
in, 142, 148 

alkalinity of, 73 
fixed, 74 
volatile, 74 

alkapton bodies in, 126 

ammonia in, 97. See also Am- 
monia in urine. 

ammoniacal decomposition, 74 

ammoniomagncsium phosphate 
crystals in, 148 

ammonium urate crystals in, 151 

amorphous phosphates in, 149 

anguillula aceti in, 171 

animal parasites in, 169 

antipyrin in, 132 

arsenic in, 133 

Gutzeit's test for, 133 
Reinsch's test for, 133 

atropin in, 133 

bacillus tuberculosis in, 168 
typhosus in, 168 

bacteria in, 72, 167 

bacterial casts in, 159 

Bence- Jones' body in, 108 
detection, 108 

bile acids in, 124 
Hay's test, 124 
tests, 124 

bile in, 123 

Gmelin's test for, 123 
Smith's test for, 123 

bile-pigment in, 123 

bilifuscin in, 123 

bilirubin in, 123 

biliverdin in, 123 



Urine, blood in, 72, 166 
blood-casts in, 158 
blood-corpuscles in, 165 
brick-dust deposit in, 72, 143 
bromids in, 133 
bubbles of air in, 173 
calcium carbonate in, 150 

oxalate in, 144 

phosphate crystals in, 149 
casts in, 152 

negative-staining, 154 
chemic examination, 80 
chlorids in, 82. See also Chlorias 

in urine. 
coffin-lid crystals in, 148 
color, 71 

composition of, 68, 80 
constituents, 68 

abnormal, 99 

inorganic, 82 

normal, 80 

organic, 82 
cryoscopy of, 79 
cylindroids in, 160 
cystin crystals in, 146 
decreased, 71 

dextrose in, 108. See also Glyco- 
suria. 
diacetic acid in, 122. See also 

Diacetic acid in urine. 
diazo substances in, 126 
dumb-bell crystals in, 150, 151 
earthy phosphates in, 149 
effect of drugs on, 71, 132 
electric conductivity, 79 
envelop crystals in, 144 
epithelial casts in, 158 

cells in, 162, 163 
examination, 70 

chemic, 80 

microscopic, 138 

physical, 70 
extraneous structures in, 171 
fat-droplets in, 172 
fat-globules in, 147 
fatty casts in, 158 
fibers in, extraneous, 161, 172 

of cotton, 161, 172 

of linen, 161, 172 

of silk, 161, 172 

of wool, 161, 172 
fibrinous casts in, 157 



INDEX 



467 



Urine, filariae in, 170 
floaters in, 169 
freezing-point, 79 
fruit-sugar in, 117 
functional tests for, 78 
glucose in, 108. See also Glyco- 
suria. 
gonococci in, 169 
gonorrheal threads in, 169 
granular casts in, 157, 158 
granule cells in, compound, 162 
gravel in, 142 
hairs in, 161 
hip-roof crystals in, 148 
hyaline casts in, 154, 155 
hyphae of molds in, 161 
in calculus, renal, 177 

vesical, 178 
in chyluria, 148 
in cystitis, 178 
in diabetes insipidus, 179 

mellitus, 179 
in disease, 173 
in hyperemia, 173, 174 
in nephritis, 173, 175, 176 
in pregnancy, 175 
in pyelitis, 177 
in renal calculus, 177 

hyperemia, 173 

tuberculosis, 175 
in vesical calculus, 178 

tuberculosis, 178 

tumors, 178 
increased, 70 
indican in, 90. See also Indican 

in urine. 
inorganic constituents, 82 
iodin in, 133 

irregular epithelial cells in, 162 
lactose in, 117 
lead in, 134 

Lederer's test, 134 
leucin in, 145 
levulose in, 117 
lycopodium granules in, 173 
- melanin in, 126 

tests for, 126 
melanogen in, 126 
mercury in, 136 
methylene-lalue test for, 79 
micrococcus ureae in, 167 
microscopic examination, 138 



Urine, milk-sugar in, 117 
mold fungi in, 172 
morphin in, 136 
mucin in, 106 
mucous threads in, 159 
normal constituents, &3 
nubecula of, 72 
odor, 73 

organic constituents, 82 
oxybutyric acid in, 123 
pavement epithelial cells in, 163 
pentoses in, 117 

Bial's orcin test, 118 
phenacetin in, 132 
phenol in, 136 
phenolphthalein in, 137 
phloridzin test for, 79 
phosphates in, 86, 148. See also 

Phosphates in urine. 
physical examination, 70 
pigments in, 71 

removal, 69 
polyhedral cells in, 162 
potassium indoxyl sulphate in, 

90. See also Indican in urine. 
proteins in, 99 
proteoses in, 106 

detection, 108 

primary, 106 

secondary, 106 
pseudocasts in, 161 
purin bodies in, 95 

Cook's method, 96 
pus in, 72, 164 

Donne's test, 72 
pus-casts in, 159 
pus-corpuscles in, 163 
quantity, 70 
quinin in, 137 
reaction, 73 
red blood-corpuscles in, 165 

sand in, 142 
resinous drugs in, 137 
retention with overflow, 70 
salicylates in, 137 
salol in, 137 
schistosomum haetaatobium in, 

170 
serum-albumin in, 99 
serum-globulin in, 99 
shadow cells in, 166 
sodium urate in, 144 



468 



INDEX 



Urine, solids in, total, 76 
Hiiser's method, 78 

specific gravity, 74 

spermatozoa in, 167 

squamous epithelial cells in, 163 

starch-granules in, 173 

sugars in, 108 

suljihates in, 88. See also 5m/- 
p/idtcs in urine. 

sul])huric acid in, 88 

sup])ression, 71 

ta.'nia echinococcus in, 169 

tannin in, 138 

thorn-apple crystals in, 151 

total solids in, 76 

Hiiser's method, 78 

transparency, 72 

trichomonas vaginalis in, 170 

triple phosphate crystals in, 148 

tube-casts in, 152 

tubercle bacilli in, 168 

tyrosin in, 145, 146 

urates in, amorphous, 72, 95, 143 
in mass, 160 

urea in, 92. See also Urea in 
urine. 

uric-acid crystals in, 142 

uric acid in, 95. See also Uric 
acid in urine. 

vinegar eel in, 171 

volatile alkalinity of, 74 

waxy casts in, 156 

yeast-cells in, 171 
Urinometer, Squibb's, 75 
Urinopyknomcter, Saxe's, 76 



Vaccine treatment, 264 
Vaccines, 419 

autogenous, 419 

bacterial, Wright's, 264 

bottles, 420 

counting of, 422 

dosage of, 425 

clinical method, 426 

in infections, 426, 427 

in malignant endocarditis, 427 

in typhoid fever, 428 

injection of, 425 
technic, 425 

method of use, 425 

preparation of, 419 



Vaccines, preparation of, diluting, 
424 
making the suspension, 421 
materials, 419 
obtaining the bacteria, 420 
sterilization, 422 
stock, 419 

therapeutic indications, 426 
Vacuolated leukocytes, 243 
Van Gieson's stain for Negri bodies, 
Frothingham's modification, 395 
Vegetable cells in feces, 316 

fibers in feces, 316 
Vermidea, 340 

Vernal catarrh, eosinophilic leuko- 
cytes in, 383 
Vesical calculus, urine in, 178 
tuberculosis, urine in, 178 
tumors, urine in, 178 
Vincent's angina, 379 
spirochaete of, 331 
Vinegar eel, 354 

in urine, 171 
Vogel's scale. See Frontispiece. 
Volume index of blood, 200 

Larrabee's method, 201 
method, 201 
Von Fleischl's estimation of hemo- 
globin, 185 
hemoglobinometer, 186 
Von Pirquet's reaction in tubercu- 
losis, 430 

Wassermann reaction, 264 
bacteriolysis in, 265 
effect of mercury treatment on, 

273 
of salvarsan treatment on, 

273 
of treatment on, 273 
hemolysis in, 265 
modifications, 271 
Noguchi's modification, 271 
reagents in, 269 
I technic, 269 

value of, 272 
Water-motor centrifuge, 84 
Watery blood, 181 
Waxy casts in urine, 156 
Weights, 439 
I Wetherill's torfuge, 139 
I Whip-worm, 364 



INDEX 



469 



White blood-corpuscles, 180 
Widal reaction, 258 

in typhoid fever, 127 

macroscopic, 261 

microscopic, 259 
Wires, platinum, 397 
Wool fibers in urine, 161, 172 
Working distance of objective, 23 
Worms, 341, 345 

eosinophilia as symptom, 239 

pin-, 355 

round-, 353 

screw-, 366 

tape-, 345 

thread-, 355 

whip-, 364 
Wright and Kinnicutt's estimation 

of blood-plaques, 216 
Wright's bacterial vaccines, 264 

blood-stain, 222 
for syphilis, 390 
in cytodiagnosis, 372 



Wright's capsule, 399 

method of obtaining blood in, 
258 
method foi measuring opsonins, 

262 
stain for malarial parasites, 254 

Xerosis bacillus in eye, 382 

Yaws, treponema pertenue of, 333 
Yeast-cells in gastric contents, 302 

in urine, 171 
Yeasts in sputum, 47 

Zappert ruling for count in leu- 
kemia, 209 

Ziehl-Neelsen method for bacillus 
tuberculosis in sputum, 51 

Zoomastigophora, 327, 330 

Zymogens, 284 



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A Complete Catalogue of our Publications will be Sent upon Request 



SAUXDEKS' BOOK'S O.V 



Musser and Kelly on 
Treatment 



JUST READY— IN THREE VOLUMES 
A Handbook of Practical Treatment. By 77 eminent 
specialists. Edited by John H. Musser, M. D., Professor of 
Clinical Medicine, University of Pennsylvania ; and A. O. J. 
Kelly, M. D., Assistant Professor of Medicine, University of 
Pennsylvania. Three octavo voknnes of about 900 pages each, 
illustrated. Per volume: Cloth, $6.00 net; Half Morocco, 

17-50 net. 

THE EMINENT CONTRIBUTORS 



Isaac A. Abt, M.D. 
Sir Clifford AUbutt, ^LD. 
James M. Anders, M.U. 
John F. Anderson, M.D. 
Le welly s F. Barker, M.D. 
Joseph C Bloodgood, M.D. 
George Bliimer, M.D. 
Sir Lauder Brunton, NLD. 
Charles W. Burr, M.D. 
Richard C. Cabot, M.D. 
James Carroll, .M.D. 
JohnG. Clark, M.D. 
Rufus L Cole, .M.D. 
Warren Coleman, M.D. 
Matthew H. Cryer, .M.D. 
Clinton T. Dent, M.D. 
Francis X. Dercum, M.D. 
Geo. E. deSchweinitz, M.D. 
George Dock, M.D. 
Isadore Dyer, M.D. 
David L. Edsall, M.D. 
William A. Edwards, M D. 
.Arthur W. Elting, M D 
John M. T. Finney, M D 
Charles H. Frazier, M.D. 
M. Howard Fussell, M.D. 



Thomas B. Futcher, M.D. 
John H. Gibbon, M.D. 
Joel E. Goldthwait, M.D. 
Samuel McC. Hamill, M.D. 
Hobart A. Hare, M.D 
Charles Harrington, M.D. 
Ludvig Hektoen, M.D. 
Albion Walter Hewlett, M.D 
Guy Hinsdale, M.D. 
Guy L. Hunner, M.D. 
Chevalier Jackson, M.D. 
Henry Jackson, M.D. 
Theodore C. Janeway, M.D. 
J. H. Jopson. .M.D. 
A. O. J. Kelly, M.D. 
Maynard I^dd. M.D. 
Egbert Lefevre, M.D. 
James Hendrie Lloyd. M.D. 
G. Hudson Makuen, M.D. 
Charles F. Martin. M.D. 
Edward Martin, M.D. 
Charles H. Mayo, M.D. 
William J Mayo, M.D. 
R. Tait McKenzie. M.D. 
Herbert C. Moffit, M.D. 
Jesse .M. Mosher, M D 



B. G. A. Moynihan, Esq. 
George P. Muller, M.D. 
John H. Musser, M.D. 
Edward O. Otis, .\LD. 
Henry K. Pancoast, M.D. 
Roswell Park, M.D. 
Richard M. Pearce, M.D. 
Charles W. Richardson, M.D. 
David Riesman, M.D. 
Milton J. Rosenau, M.D. 
Joseph Sailer, ALD. 
J. F. Schamberg, M.D. 
Henry Sewell, .M.D. 
Bertram W. Sippy, M.D. 
William G. Spiller, M D. 
J. Dufton Steele. ALD. 
Alfred Stengel, M.D. 
Charles G. Stockton, .M.D. 
James E. Talley, M.D. 
E. W. Taylor, M.D. 
James Tyson, M.D. 
George H. Weaver, M.D. 
J. William White, M.D. 
AlfredC. Wood, M.D. 
Horatio C. Wood, Jr., M.D. 



DIAGNOSIS AND TJiEATMENT 



Cabot's Differential Diag'nosis 

Differential Diagnosis. Presented through an analysis of 
383 Cases. By Richard C. Cabot, M.D., Assistant Professor of 
Clinical Medicine, Harvard Medical School, Boston. Octavo of 
753 pages, illustrated. Cloth, ^5.50 net. 

THREE PRINTINGS IN FOUR MONTHS 

Dr. Cabot's work takes up diagnosis from the point of view of the pre- 
senting symptom — tlie symptom in any disease which holds the foreground in 
the clinical picture : the principal complaint. It groups diseases under these 
symptoms, and points the way to proper reasoning in coming to a correct 
diagnosis. It works backward from each leading symptom to the actual 
organic cause of the symptom. 



Morrow's Diagnostic and 
Therapeutic Technic 

Diagnostic and Therapeutic Technic. By Albert S. 
Morrow, M.D., Adjunct Professor of Surgery, New York Poly- 
clinic. Octavo of 775 pages, with 815 original line drawings. 
Cloth, $5.00 net. 

JUST THE WORK FOR PRACTITIONERS 

Dr. Morrow's new work is decidedly a work for you — the physician en- 
gaged in general practice. It is a work you need because it tells you just 
how to perform those procedures required of you every day, and it tells you 
and shows you by clear, «««/ line-drawings, in a way never before approached. 
.The information it gives is such as you need to know every day — transfusion 
and infusion, hypodermic medication, Bier's hyperemia, collection and 
preservation of pathologic material, exploratory punctures, aspirations, anes- 
thesia, etc. Then follow descriptions of those measures employed in the 
diagnosis and treatment of diseases of special regions or organs. 



SAUNDE/iS' BOOK'S OX 



Bonney on Tuberculosis 



Tuberculosis. By Sherman G. Bonney, M. D., Professor of 
Medicine, Denver and Gross College of Medicine, Denver. Octavo 
of 955 pages, with 243 original illustrations. Cloth, $7.00 net. 

THE NEW (2d) EDITION 

Dr. Honney's work is a thorough and complete treatise of the entire sub- 
ject of tuberculosis. The section on Physical Signs of Pulmonary Tuber- 
culosis is really a complete monograph on the physical diagnosis of diseases 
of the chest. Treatment is particularly full and practical. There are chapters 
on prophylaxis ; open-air treatment, fully illustrated ; diet ; sanitarium and 
climatic treatment ; therapeutic measures to alleviate distressing symptoms; 
and drug and vaccine therapeutics. 

Maryland Mediczd Journal 

•■ Dr. Bonney 's book is one of the best and most exact works on tuberculosis, in all ils 
aspects, that has yet been published." 

Anders and Boston's Medical 
Diagnosis 

A Text-Book of Medical Diagnosis. By James M. An- 
ders, M.D., Ph.D., LL. D., Professor of the Theory and Prac- 
tice of Medicine and of Clinical Medicine, and L. Napoleon 
Boston, M.D., Adjunct Professor of Medicine, Medico-Chirur- 
gical College, Philadelj)hia. Octavo of 11 75 pages, with 443 
illustrations. Cloth, $6.00 net. 

JUST READY 

This new work is tlesigned expressly for the general practitioner. The 
methods given are practical and es^>ecially adapted for quick reference. The 
diagnostic methods are presented in a forceful, definite way by men who have 
had wide experience at the bedside and in the clinical laboratory. The text 
IS profusely illustrated with original pictures, each one representing some 
point in technic or some diagnostic sign. It is a work for every practitioner. 



PRACTICE OF AfEDlCtNE 



Kemp on 
Stomach and Intestines 



Diseases of the Stomach and Intestines. By Robert 
Coleman Kemp, M. D., Professor of Gastro-intestinal Diseases 
at the New York School of Clinical Medicine. Octavo of 766 
pages, with 279 illustrations. Cloth, $6.00 net. 

FOUR PRINTINGS IN FIFTEEN MONTHS 

It is the practitioner who first meets with these cases, and it is he upon 
whom the burden of diagnosis rests. After the diagnosis is established, the 
practitioner, if properly equipped, could frequently treat the case himself 
instead of transferring it to a specialist. This work is intended to equip the 
practitioner with this end in view: 

The Therapeutic Gazette 

" The therapeutic advice which is g^ven is excellent. Methods of physical and 
chemical examination are adequately and correctly described." 



Deaderick on Malaria 



Practical Study of Malaria. By William H. Deaderick, 
M. D., Member American Society of Tropica) Medicine. 
Octavo of 402 pages, illustrated. Cloth, $4.50 net; Half 
Morocco, ^6.00 net. 

A WORK LONG NEEDED 

This is the only book in any language describing the third cycle of the 
malarial parasite — the parthenogenetic cycle. The chapters on diagnosis 
and treatment are conspicuous for the exactness of statement and the intuitive 
way in which the author has grasped the needs of the physician and supplied 
them. 

Frank A. Jones. «M. D., 

Professor of Clinical Medicine and Physical Diagnosis, Memphis Hospital Medical 
College. 

" Dr. Deaderick's book is up to date and the subject matter is well arranged. We 
have been waiting for many years for such a work written by a man who sees malaria in 
all its forms in a highly malarious climate." 



SAUNDERS' BOOKS ON 



Tousey's 

Medical Electricity anZ X-Rays 

Medical Electricity and the X-Rays. By Sinclair 
TousEY, M. D., Consulting Surgeon to St. Bartholomew's 
Hospital, New York. Octavo of 1116 pages, with 750 illustra- 
tions, 16 in colors. Cloth, $7.00 net. 

ADOPTED BY THE U. S. ARMY 

This new work by such an eminent authority is destined to take a leading 
place among books on this subject. Written primarily for the general prac- 
titioner, it gives him just the information he wishes to have regarding the use 
of medical electricity, the therapeutic results obtained, etc. At the same time 
it tells the specialist how the most eminent electiotherai)eutists are securing 
results, the latest authorities in every country having been consulted for de- 
tails of practical value. The work gives explicit directions for the care and 
regulation of static machines, x-ray tubes, and all apparatus. The author 
tells how to make x-ray pictures by a practical technic easily followed, even 
though the operator be inexperienced in this field. Being an 'authority on 
dental radiography, the chapters on this side of the subject are particularly 
imp>ortant to those interested in dental work. 
The Therapeutic Gazette 

" Dr. Tousey's book may be said to contain practically everything in regard to medical 
electricity, and by ' everything ' we mean not only a discussion of elementary facts, largely 
physical in nature, but a description of all the forms which can be employed." 

McKenzie on Exercise in 
Education and Medicine 

Exercise in Education and Medicine. By R. Tait 
McKenzie, B. A., M. D., Professor of Physical Education, and 
Director of the Department, University of Pennsylvania. Oc- 
tavo of 406 pages, with 346 illustrations. Cloth, $3.50 net. 

ILLUSTRATED 

This work is a full and detailed treatise on the application of systematized 
exercise in the development of the normal body and in th# correction of cer- 
tain diseased conditions in which gymnastics have proved of value. 

D. A. Sar^eantt M. D., Director of Hemenivay Gymnasium, Har^iard University. 

" It cannot fail to be helpful to practitioners in medicine. The classification of athletic 
games and exercises in tabular form for different ages, sexes, and occupations is the work of 
an expert. It should be in the hands of every physical educator and medical practitioner." 



THE PRACTICE OF MEDICINE 



Anders* 
Practice of Medicine 

A Text-Book of the Practice of Medicine. By James 
M. Anders, M. D., Ph. D., LL. D., Professor of the Practice 
of Medicine and of Clinical Medicine, Medico-Chirurgical Col- 
lege, Philadelphia. Handsome octavo, 1326 pages, fully illus- 
trated. Cloth, $5.50 net; Half Morocco, $7.00 net. 

THE NEW (9th) EDITION 

The success of this work is no doubt due to the extensive consideration 
given to Diagnosis and Treatment, under Differential Diagnosis the jxiints of 
distinction of simulating diseases being presented in tabular form. In this 
new edition Dr. Anders has included all the most important advances in 
medicine, keeping the book within bounds by a judicious elimination of 
ol'solete matter. A great many articles have also been rewritten. 

Wm. E. Quine, M. D., College of Physicians and Surgeons, Chicago. 

" I consider Anders' Practice one of the best single-volume works before the profession 
at this time, and one of the best text-books for medical students." 



DaCosta's Physical Diagnosis 

Physical Diagnosis. By John C. DaCosta, Jr., Asso- 
ciate in Clinical Medicine, Jefferson Medical College. Octavo 
of 557 pages, with original illustrations. Cloth, $3.50 net. 

ORIGINAL ILLUSTRATIONS 

In Dr. DaCosta's work every method given has been carefully tested and 
proved of value by the author himself. Normal physic.il signs are explained 
in detail in order to aid the diagnostician in determining the abnormal. Both 
direct and differential diagnoses are emphasized. The 212 original illustra- 
tions are artistic as well as practical. 

Henry L. Eisner, M. D., Professor of Medicine, Syracuse University. 

" I have reviewed this book and am thoroughly convinced that it is one of the best 
ever written on the subject. In every way I find'it a superior production." 



SAUNDERS' BOOKS ON 



Sahli's Diag(nostic Methods 

Edited by Nath'l Bowditch Potter. M.D. 



A Treatise on Diagnostic Methods of Examination. 

By Prof. Dr. H. Sahli, of Bern. Edited, with additions, by 
Nath'l Bowditch Potter, M.D., Assistant Professor of Clinical 
Medicine, Columbia University. Octavo of 1225 pages, pro- 
fusely illustrated. Cloth, $6.50 net. 

THE NEW (2d) EDITION, RESET 
Lewellys F. Barker. M. D. 

Professor of Medicine, Johns Hopkins UniTersHy 
" 1 am delighted with it, and it will be a pleasure to recommend it to our students in 
the Johns Hopkins Medical School." 

Friedenwald and Ruhrah 
on Diet 



Diet in Health and Disease. By Julius Friedenwald, 
M. D., Professor of Diseases of the Stomach, and John Ruhrah, 
M. D., Professor of Diseases of Children, College of Physicians 
and Surgeons, Baltimore. Octavo of 764 pages. Cloth, 
$4.00 net. 

NEW (3d) EDITION 

This work contains a complete account of foodstuffs, their uses, and 
chemical composition. Dietetic management in all diseases in which diet 
plays a part in treatment is carefully considered, the articles on diet in diseases 
of the digestive organs containing numerous diet-lists and explicit instructions 
for administration. The feeding of infants and children, of patients before 
and after anesthesia and surgical opention.s, are all taken up in detail. 

George Dock. M. D., 

Professor of Theory and Practice of Medicine and Clinical Medicine, Tulane Uni- 
versity of Louisiana. 

" It seems to me that you have prepared the most valuable work of the kind now avail- 
able. I am especially glad to see the long list of analyses of different kinds of food " 



PRACTICE OF MEDICINE. 



Oertel on Brig'ht's Disease 

The Anatomical and Histological Processes of Bright 's 
Disease. By Horst Oertel, M. D., Director of the Riissell 
Sage Institute of Pathology, New York. Octavo of 227 pages, 
with 44 text-illustrations and 6 colored plates. Cloth, $5.00 net. 

ILLUSTRATED 

These lectures deal with the anatomic and histologic processes of Bright' s 
disease, and in a somewhat different way from the usual manner. Everywhere 
relations are emphasized and an endeavor made to reconstruct the whole as a 
unit of interwoven processes. In the preparation of his lectures the author 
had in mind a twofold aim : To present the visual picture of nephritis, and 
to prepare the proper way for the understanding of the genesis of the disease. 



Fenwick on Dyspepsia 

Dyspepsia. By William Soltau Fenwick, M. D., of Lon- 
don. Octavo of 485 pages, illustrated. Cloth, $3.00 net. 
Southern Medical Journal 

*' The suggestions on treatment are logical and practical, being particularly helpful in 
many of those perplexing types so often encountered." 



Smith's What to Eat 6 Why 

What to Eat and Why. By G. Carroll Smith, M.D., 
Boston. i2mo of 312 pages. Cloth, $2.50 net. 

RECENTLY ISSUED 

With tliis book you no longer need send your patients to a specialist to 
be dieted — you will be able to prescribe the suitable diet yourself, just as you 
do other forms of therapy. Dr. Smith gives "the why" of each statement 
he makes. It is this knowing wliy which gives you contidence in the book, 
which makes you feel that Dr. Smith knows. 



Slade's Physical Examination O Diagnostic Anatomy 

Physical Ex.'vmination Axn Dia(;N(«tic Anatomy. — By Charm. s 
B. Sladk, M.D., Chief of Clinic in General Medicine, University and 
Bellevue Hospital Medical College. i2mo of 146 pages, illustrated. 
Cloth, $1.25 net. 



lo SAUNDERS' BOOKS ON 

AMERICAN EDITION 

NOTHNAGEL'S PRACTICE 

UNDER THIi EDITORIAL SUI'EUVISION OK 

ALFRED STENGEL. M.D. 

Professor of Medicine in the University of Pennsylvania 



Typhoid and Typhus Fevers 

\^y I)K. II. CURSCHMANN, of Leipsic. Edited, with additions, by 
William Oslkr, M. D., F. R. C. P., Oxford University, Oxford, 
England. Octavo of 646 pages, illustrated. 

Sm&llpox (including Vaccination) , Varicella, Cholera 
Asiatica, Cholera Nostras, Erysipelas, Erysip- 
eloid, Pertussis, and Hay Fever 

By Dr. H. Immermann, of Uasle ; I)k. Th. von JCkcensen, of 
Tubingen ; Dr. C. Lieiskkmeistkr, of Tubingen ; Dr. II. Lenhartz, 
of Hamburg; and Dr. G. Sticker, of Giessen. The entire volume 
edited, with additions, by Sir J. \\. Moore, M. D., F. R. C. P. I., 
Royal College of Surgeons, Ireland. Octavo of 682 pages, illustrated. 

Diphtheria, Measles, Scarlet Fever, and Rotheln 

By William P. Northrup, M. D., of New York, and Dr. Th. 
von JOrgensen, of Tubingen. The entire volume edited, with additions, 
by William P. Northrup, M. D., University and Bellevue Hospital 
Medical College. Octavo of 672 pages, illustrated. 

Diseases of the Bronchi, Diseases of the Pleura, and 
Inflanmations of the Lunges 

By Dr. F. A. Hoffmann, of Leipsic; Dr. O. Rosenbach, of 
Berlin; and Dr. F. Ai'FRECHT, of Magdeburg; The entire volume 
edited, with additions, by John H. Musser, M. D., University of Penn- 
sylvania. Octavo of 1029 pages, illustrated. 

Diseases of the Pancreas, Suprarenals, and Liver 

By Dr. T. Oser, of Vienna; Dr. E Nitsskr. of Vienna; and Drs. 
IT. Quincke and G. Hoppe-Sevi.er, of Kiel. The entire volume 
edited, with additions, by Recinald H. Fit/., A. M., M. D., Harvard 
University; and Frederick A. Packard, M. D., Pennsylvania and 
Children's Hospitals, Philadelphia. Octavo of 918 pages, illustrated. 

PER VOLUME: CLOTH, $5.00 NET; HALF MOROCCO. $6.00 NET 



PRACTICE OF MEDICINE ll 

AMERICAN EDITION 

NOTHNAGEL'S PRACTICE 



Diseases of the Stomach 

By Dr. F. Riegil, of Giessen. Edited, with additions, by Charles 
G. Stockton, M. D., University of Buffalo. Octavo of 835 pages. 

Second 

Diseases of the Intestines and Peritoneum Edition 

By Dr. Hermann Nothnagel, of Vienna. Edited, with additions, 
by H. D. R0LI.ESTON, M. D., F. R. C. P., St. Georges Hospital, 
London. Octavo of 1 100 pages, illustrated. 

Tuberculosis and Acute General Miliary Tuberculosis 

By Dr. G. Cornet, of Berlin. Edited, with additions, by Wai.ier 
B. J.AMES, M. D., Columbia University, New York. Octavo of 806 pages. 

Diseases of Blood {Anemia, Chlorosis, Leukemia, Pseudoleukemia) 

By Dr. P. Ehrlich, of Frankfort-on-the-Main ; Dr. A. Lazarus, of 
Charloitenburg ; Dr. K. von Noorden, of Frankfort-on-the-Main; and 
Dr. Felix Pinki;s, of Berlin. The entire volume edited, with addi- 
tions, by Alfred Stengel, M. D., University of Pennsylvania. Octavo 
of 714 pages, illustrated. 

Malaria, Influenza, and Dengue 

By Dr. J. Mannaberg, of Vienna, and Dr. O. Leichtenstern, of 
Cologne. The entire volume edited, with additions, by Ro.nald Ro.ss, 
F. R. C. S., University of Liverpool ; J. W. \V. Stephens, M. D., 
D. P. H., University of Liverpool ; and Albert S. Grunbaum, F. 
R. C. P., University of Liverjxjol. Octavo of 769 pages, illustrated. 

Kidneys, Spleen, and Hemorrhagic Diatheses 

By Dr. H. Senator, of Berlin, and Dr. ^L Litten, of Berlin. The 
entire volume edited, with additions, by James B. Herkick, M. D., 
Rush Medical College. Octavo of 815 pages, illustrated. 

Diseases of the Heart 

By Prof. Dr. Th. von JCrgensen, of Tubingen ; Prof. Dr. L. 
Krehl, of Griefswald; and Prof. Dr. L. von Schrotter, of 
Vienna. The entire volume edited, with additions, by George Dock, 
M. D., Tulane University of Louisiana. Octavo of 848 pages. 

- PER VOLUME: CLOTH. $5.00 NET; HALF MOROCCO, $6.00 NET 



Goepp's State Board Questions 

State Bo.-vrd Questions and Answers. By R. Max Goepp, 
M. D., Professor of Clinical Medicine, Philadelphia Polyclinic. Octavo 
of 715 pages. Second Edition. Cloth, ^(4.00 net. 

" Nothing has been printed which is so admirably adapted as a guide and self-quiz 
for those intending to take State Board Examinations." — Pennsylvania Medical 
Journal 



SAUNDERS' BOOKS ON 



Stevens* Therapeutics New (5th) Edition 

A Text-Book ok Modern Materia Medica and Therapeutics. 
Hy A. A. Stevens, A.M., M.D., Lecturer on Physical Diagnosis in the 
University of Pennsylvania. Octavo of 675 pages. Cloth, ^3.50 net. 

Dr. Stevens' Therapeutics is one of the most successful works on the subject ever 
published. In this new edition the work has undergone a very thorough revision, 
and now represents the very latest advances. 

The Medical Record, New York 

'* Among the numerous treatises on this most important branch of medical practice, 
this by Dr. Stevens has ranked with the best." 

Butler's Materia Medico New (6th) Edition 

A Text-Book of Materia Medica, Therapeutics, and Pharma- 
cology. By George Y. Buti.er, Ph.G., M.I)., Professor and Head 
of the Department of Therapeutics and Professor of Preventive and 
Clinical Medicine, Chicago College of Medicine and Surgery, Medical 
Department V^alpariso University. Octavo of 702 pages, illustrated. 
Cloth, ;g4.oo net ; Half .Morocco, ;$5. 50 net. 

For this sixth edition Dr. Butler has entirely remodeled his work, a great part hav- 
ing been rewritten. All obsolete matter has been eliminated, and special attention 
has been given to the toxicologic and therapeutic effects of the newer compounds. 

Medical Record, New York 

" Nothing has been omitted by the author which, in his judgment, would add to 
the completeness of the text." 

SoUmann's Pharmacology New (2d) Edition 

A Text-Book of Pharmacology. By Torald Sollmann, M.D., 
Professor of Pharmacology and Materia Medica, Western Reserve Uni- 
versity. Octavo of 1070 pages, illustrated. Cloth, $4.00 net. 

The author bases the study of therapeutics on systematic knowledge of the nature 
and properties of drugs, and thus brings out forcibly the intimate relation between 
pharmacology and practical medicine. 

J. F. Fotherin^ham, M.D., Trinity Medical College, Toronto. 

" The work certainly occupies ground not covered in so concise, useful, and scien- 
tific a manner by any other text 1 have read on the subjects embraced." 

Arny*s Pharmacy 

Principles of Pharmacy. By Henry V. Arny, Ph. G., Ph. D., 
Professor of Pharmacy at the Cleveland School of Pharmacy. Octavo of 
"75 P^g^s, with 246 illustrations. Cloth, $5.00 net. 

George Reimann, Ph. G., Secretary of the Ne7v York state Board 0/ Pharmacy. 

" I would say that the book is certainly a great help to the student, and I think it 
ought to be in thehands of every person who is contemplating the study of pharmacy." 



THERAPEUTICS AND MATERIA MEDICA 13 



Hinsdale's Hydrotherapy 

Hydrotherapy : A Treatise on Hydrotherapy in General ; 
Its Application to Special Affections; the Technic or Processes 
Employed, and a Brief Chapter on the Use of Waters Internally. 
By Guy Hinsdale, M.D., Fellow of the Royal Society of Great 
Britain. Octavo of 466 pages, illustrated. Cloth, $3.50 net. 

The treatment of disease by hydrotheiapeutic measures has assumed such 
an important place in medical practice that a good, practical work on the 
subject is an essential in every practitioner's armamentarium. This new 
work supplies all needs. It describes fully the various kinds of baths, douches, 
sprays ; the application of heat and cold ; the internal use of mineral waters 
and all other procedures included under hydrotherapeutic measures. Then 
the use of hydrotherapy in the various diseases is detailed concisely. 



Kelly's Cyclopedia of American 
Medical Biography 

Cyclopedia of American Medical Biography. By How- 
ard A. Kelly, M. D., Professor of Gynecologic Surgery at Johns 
Hopkins University. Two octavos of 750 pages each, with por- 
traits. 

JUST READY 

Dr. Kelly, in these two handsome volumes, presents concise, yet com- 
plete biographies of tliose men and women who have contributed notewor- 
thily to the advancement of medicine in America. Dr. Kelly's reputation for 
painstaking care assures accuracy of statement. There are about one thousand 
biographies included. 

Swan's Prescription-writing and Formulary 

Prescription WRiriNG and Formul.ary. By John M. Swan, 
M.D., Director Glen Springs .S*iitarium, Watkins, N. Y. l2mo of 185 
pages. Flexible cloth, $1.25 net. 

Stewart's Pocket Therapeutics and Dose- 
book New (4th) Edition 
Pocket Therapei:tics and Dose-book. By Morse Siewart, Jr., 
M.D. 32mo of 263 pages. Cloth, ;$i.oo net. 



14 SAUNDERS' BOOKS ON 

THE BEST /iinerican standard 

Illustrated Dictionary 

Just Ready — The New (6th) Edition, Reset 

The American Illustrated Medical Dictionary. By W. A. 

Nkw.man Dokland, M. D., Editor of "The American Pocket 
Medical Dictionary." Octavo of 975 pages. Flexible leather, 
54.50 net; with thumb index, $5.00 net. 

A NEW WORK— WITH ADDED FEATURES 

Howard A. Kellyt M. D.^ Johns Hopkins University, Baltimore. 

" Dr. Borland's dictionary is admirable. It is so well gotten up and of such conve- 
nient size. No errors have been found in my use of it." 



Thornton's Dose-Book Fourth EdMon 

Dose-Book and Mamai. of PRKSCRiprioN-WRiTiNG. By E. Q. 

Thornton, M. D., .Assistant Professor of Materia Medica, Jefferson 

Medical College, Philadelphia. Post-octavo, 392 pages, illustrated. 

Flexible leather, S2.00 net. 

" It will afford me much pleasure to recommend the book to my classes, who often 
fail to find such information in their other lexu-books." — C. H. Miller, M.D., 
Profi-ssor of l^hatmacology. Northwestern University Meiiical School, Chicago. 

Lusk on Nutrition New (2d) Edition 

Elements of the Science of Nutrition. By Graham Lusk, 
Ph.D., Profes.-or of Physiology. in Cornell University Medical School. 
Octavo of 402 pages. Cloth, J3.00 net. 

"I shall recommend it highly. It is a comfort to have such a discussion of the 
subject."— Lewkllvs K. Hakkek, M. D., Pro/etsor 0/ the PrincipUt and Practice 
0/ Meclicine, Johns Hopkins University. 

Hatcher and Sollmann's Materia Medica 

A 1 E.KT-BooK OF M.VFERIA Medica : including Laboratory Exer- 
cises in the Hi.stologic and Chemic E.xamination cf Drugs. By Robert 
A. n.ATCHER, Ph. (j., M. D. ; and ToRALD Soi.lmann, M. D. i2mo 
of 4 1 1 pages. Flexible leather, $2 00 net. 

Bridi^e on Tuberculosis 

Tuber( ui.osis. By Norman Bridge, A. M., M. D, i2mo of 302 
pages, illustrated. Cloth, ^l .50 net. 



MATERIA MEDICA AMD THERAPEUTICS. 15 

American Pocket Dictionary ^,^ JS;)^^Z 

The American Pocket Medical Dictionary. Edited by W. 
A. Newman Borland, M.D. Flexible leather, with gold edges, $1 00 
net ; with thumb index, $1.25 net. 

Eichhorst's Practice of Medicine 

A Text-Book of the Practice of Medicine. By Dr. H. Eich- 
HORST, University of Zurich. Edited by A. A. Eshnkr, M. D. Two 
octavos of 6oo pages each, illustrated. Per set : Cloth, ;$6.oo net. 

Pusey and Caldwell on X-Rays Second Edition 

The Practical Application of the Rontgen Rays in Thera- 
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